High pH protein refolding methods

ABSTRACT

Provided herein are methods for refolding denatured protein (e.g., from inclusion bodies) that do not require the use of a denaturing agent. Exemplary methods use a high pH for solubilizing denatured protein, followed by a decrease in pH for refolding the proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of International Application No.PCT/US2014/015702, filed Feb. 11, 2014, which claims priority to U.S.provisional application 61/763,664, filed on Feb. 12, 2013, the contentsof both of which are specifically incorporated by reference herein.

BACKGROUND

The backbone of an antibody, known as the Fc region, is responsible forpharmacokinetic properties that may be desirable in the case of manytherapeutic biologics (Jeffries, B. Biotechnol Prog. 2005; 21: 11-16).The size of the Fc region makes it resistant to renal filtration andbinding to the Fc Neonatal Receptor (FcRn) allows it to escape endosomaldegradation by a recycling mechanism. In addition to the Fc region thatis present in monoclonal antibody therapeutic products, there are Fcfusion products being investigated and developed (Hakim et al. Mabs.2009; 1:281-287). Fc fusions are the fusion of an Fc region to anotherprotein, peptide, or Active Pharmaceutical Ingredient (API). The Fcfusion then has both the properties of the Fc region and the therapeuticproperties of the API.

There are many cell lines that are capable of being used to manufacturetherapeutic biologics (Jung et al. Curr Opin Biotechnol. 2011; 22:1-10).The mammalian Chinese Hamster Ovary (CHO), insect Sf9, yeast S.cereviae, and bacterial E. coli are some of the most common cell linesthat are discussed for recombinant protein production. So far, yeast,CHO and E. coli have been used for manufacture of Fc containingtherapeutic biologics, including a large number of monoclonalantibodies. Expression in E. coli offers three potential and significantadvantages over expression in other cell lines: the cell linedevelopment time is much shorter; the bioreactor runs are up to 7-foldshorter, resulting in a lower capital investment; and there is no needto control aberrant glycosylation that can occur in yeast and mammaliancell cultures.

Expression of larger proteins, like Fc fusions, in E. coli can be aunique challenge. E. coli lack the chaperone proteins and otherrefolding machinery found in a eukaryotic expression system. Thecytoplasm of E. coli is also a reducing environment, which is notfavorable for the formation of disulfide bonds. The Fc region of humanIgG1 antibodies contains six disulfide bonds. Two disulfide bonds thehinge region join two peptide chains to form the homodimeric moleculeand there are two more disulfide bonds within each of the peptidechains. E. coli also have a mechanism to prevent unfolded proteins frominterfering with normal cell processes. Unfolded protein is shunted andisolated in insoluble aggregates, called Inclusion Bodies (IB), whichcan then be isolated in the insoluble fraction following cell lysis.Alternatively, when the rate of recombinant protein production is slowedto allow the protein to fold, a leader sequence may be added to directsoluble protein that is expressed to the periplasmic space. Theperiplasm is an oxidative environment favorable for the formation ofdisulfide bonds. However, the reported expression levels of recombinantprotein in the periplasm remain low (Liu et al. Protein ExpressionPurif. 2008; 62:15-20).

In contrast, E. coli expression levels in IBs have been reported to behigh. Expressing protein in IBs also has the advantages of resistance toprotein degradation, and ease of isolation from the cells (Grune et al.Int J Biochem Cell Biol. 2004; 36:2519-2530). Since an IB is aninsoluble aggregate, there may be a challenge in restoring the proteinof interest to its biologically active conformation (Jungbauer et al. JBiotechnol. 2006; 587-596). Typically, a process is required to breakapart and solubilize the IB. Then the protein must be renatured, orrefolded, into the biologically active conformation while minimizinglosses due to aggregation and precipitation. Current refolding processesmay be specific to a given protein, requiring thorough optimization foreach case. Many refolding processes require very low proteinconcentrations and consequently large volumes for the operation. This isdifficult because it requires a larger amount of potentially expensivereagents. There is also a challenge in a manufacturing setting, wherethere is a physical limit to the container size that may be used torefold proteins. Finally, in the case of Fc fusions, the refoldingprocess must correctly form the six disulfide bonds that exist in thenative form of the protein.

SUMMARY

Provided herein are methods for refolding a denatured protein,comprising, e.g., (i) suspending a denatured protein in a suspensionsolution to obtain a composition comprising suspended denaturedproteins; (ii) combining the composition comprising suspended denaturedproteins with a solubilization buffer having a pH in the range of 10.5to 13 to thereby obtain a composition comprising solubilized denaturedproteins; and (iii) combining the composition comprising solubilizeddenatured proteins with a refold buffer having a pH in the range of 9 to11 to thereby obtain a composition comprising refolded proteins. Incertain methods, the method does not include the use of a significantamount of denaturing agent and/or reducing agent. The pH of thesolubilization buffer may be in the range of pH 11.5 to 12.8, such as inthe range of pH 12.0 to 12.6. The pH of the refold buffer may be in therange of pH 10 to 10.6, such as in the range of pH 10.3 to 10.5. Thesuspension solution may consist of water. The composition comprisingsolubilized denatured protein may have a pH in the range of 11 to 13,such as a pH in the range of pH 11.5 to 12.8, e.g., pH 12.0 to 12.6. Thecomposition comprising refolded protein may have a pH in the range of 10to 11, such as in the range of 10 to 10.6, e.g., pH 10.3 to 10.5.

The denatured proteins may be suspended in suspension solution at aratio of weight (g) of denatured proteins:volume (ml) of suspensionsolution of 1:1-3, e.g., about 1:2. The suspension solution may bewater. The composition comprising suspended denatured proteins may becombined with solubilization buffer at a ratio of weight (g; e.g.,weight prior to adding suspension solution) of denatured proteins orvolume of suspension solution (ml):volume (ml) of solubilization bufferof 1:10-30, such as about 1:20. The composition comprising solubilizeddenatured proteins may be combined with refold buffer at a ratio ofvolume of solubilization buffer:volume of refold buffer of 1:1-5, suchas about 1:3-4.

In the methods described herein, the denatured proteins may be suspendedin suspension solution at a ratio of weight (g) of denaturedproteins:volume (ml) of suspension solution of 1:1-3; the compositioncomprising suspended denatured proteins may be combined withsolubilization buffer at a ratio of weight (g) of denaturedproteins:volume (ml) of solubilization buffer of 1:10-30; and thecomposition comprising solubilized denatured proteins may be combinedwith refold buffer at a ratio of volume of solubilization buffer:volumeof refold buffer of 1:1-5. The solubilization buffer may have a pH inthe range of 11.5 to 12.8 and the refold buffer may have a pH in therange of 10 to 10.9. The denatured proteins may be suspended insuspension solution at a ratio of weight (g) of denaturedproteins:volume (ml) of suspension solution of about 1:2; thecomposition comprising suspended denatured proteins may be combined withsolubilization buffer at a ratio of weight (g) of denaturedproteins:volume (ml) of solubilization buffer of about 1:20; and thecomposition comprising solubilized denatured proteins may be combinedwith refold buffer at a ratio of volume of solubilization buffer:volumeof refold buffer of about 1:3-4. The denatured proteins may be suspendedin water at a ratio of weight (g) of denatured proteins:volume (ml) ofsuspension solution of about 1:2; the composition comprising suspendeddenatured proteins may be combined with solubilization buffer having apH of about 12.2 at a ratio of weight (g) of denatured proteins:volume(ml) of solubilization buffer of about 1:20; and the compositioncomprising solubilized denatured proteins may be combined with refoldbuffer having a pH in the range of 10.2 to 10.6 at a ratio of volume ofsolubilization buffer:volume of refold buffer of about 1:3-4.

In the methods described herein, the suspended denatured proteins andthe solubilization buffer may be combined for 1-10, e.g., 2-5, minutesprior to being combined with the refold buffer. The compositioncomprising the solubilized denatured proteins may be combined with therefold buffer for 5-60, e.g., 15-25, minutes.

In the methods described herein, the pH of the solution comprising therefolded proteins may be reduced following refolding.

In the methods described herein, the solubilization buffer and/or therefold buffer may comprise Arginine. The refold buffer may comprise anoxidizing agent, e.g., glutathione, wherein, e.g., glutathione is at anabout 5:1 oxidized:reduced ratio.

In certain embodiments, the method does not comprise first suspendingthe denatured protein in a suspension solution. In certain embodiments,the method does not include the use of a denaturing agent. The denaturedproteins may be in the form of inclusion bodies (IBs). The protein thatis renatured according to the methods described herein may comprise atleast one cysteine. The protein may comprise at least two cysteines thatform a disulfide bond in the native protein. The protein may comprise anFc region, which may comprise a hinge. The protein may comprise abinding domain that specifically binds to a target protein. The bindingdomain may be an alternative scaffold binding domain, such as afibronectin based scaffold domain, e.g., a ¹⁰FN3 domain. In certainembodiments, the protein comprises a ¹⁰FN3 protein and an Fc regioncomprising a hinge, a CH2 and a CH3 domain.

Also provided herein are compositions, e.g., compositions comprising aprotein comprising at least two cysteines that form a disulfide bondunder appropriate conditions, and water, wherein the composition doesnot comprise a buffer or a denaturing agent. Also provided arecompositions comprising a suspension of denatured proteins, wherein atleast some proteins comprise at least two cysteines that form adisulfide bond under appropriate conditions, and wherein the compositiondoes not comprise a buffer or a denaturing agent. Also provided arecompositions comprising a protein comprising at least two cysteines thatform a disulfide bond under appropriate conditions, and a solubilizationbuffer having a pH in the range of pH 10 to 13, wherein the compositiondoes not comprise a denaturing agent. Further provided are compositionscomprising a protein comprising at least two cysteines that form adisulfide bond under appropriate conditions, and a refold buffer havinga pH in the range of pH 9 to 11 and an oxidizing agent, wherein thecomposition does not comprise a reducing agent other than a reducingagent that part of an oxidizing agent that is present in thecomposition. The protein may comprise an Fc region or a portion thereof.The protein may comprise a binding domain, e.g., an FBS domain, e.g., a¹⁰Fn3 domain. The ¹⁰Fn3 domain may bind specifically to a target, andthe ¹⁰Fn3 domain may comprise an amino acid sequence that is at least50% identical to any of SEQ ID NOs: 1-29. The protein may be present inthe composition at a concentration of at least 5 mg/ml or 10 mg/ml.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a diagram showing exemplary steps for refolding a denaturedprotein.

FIGS. 2A-2C show the results of the use of a G25 buffer exchange methodto estimate refold efficiency. FIG. 2A) G25 refold performed with 50 mMTris pH 8.5 and 0.4 M Arginine. FIG. 2B) G25 refold performed with 50 mMTris pH 9.0 and 0.4 M Arginine. FIG. 2C) G25 refold performed with 50 mMTris pH 10.4 and 0.4 M Arginine.

FIGS. 3A and 3B show soluble aggregate concentration visualized bySDS-PAGE. SDS PAGE analysis of G25 refolds performed at pH 9.0 (FIG. 3A)and pH 10.4 (FIG. 3B).

FIGS. 4A and 4B show SDS-PAGE gels of G25 refold reactions incubated for30 minutes (FIG. 4A) or 4 hours (FIG. 4B) in the absence of TCEP priorto loading on SDS-PAGE.

FIG. 5 shows dimer formation efficiency after 0, 1 and 2 hour refoldincubation periods prior to redox addition. “Dil” refers to the lowerconcentration of ¹⁰Fn3/Fc relative to the concentration of the samplesreferred to as “neat.”

FIGS. 6A and 6B show Near and Far UV Circular Dichroism (CD) of ¹⁰Fn3/Fcin different conditions. FIG. 6A shows Near UV CD representing tertiarystructure of the protein at varying pHs or in the presence of guanidine.FIG. 6B shows Far UV CD representing secondary structure of the proteinat varying pHs and in the presence of guanidine.

FIG. 7 shows high pH solubilization for refolding ¹⁰Fn3/Fc at 1.75, 3.5and 7 mg/ml with either a 10 μg load (lanes 1-4) or 20 μg load (lanes5-7).

FIGS. 8A and 8B show SPR binding data of (FIG. 8A) mammalian expressedand (FIG. 8B) E. coli expressed and refolded ¹⁰Fn3/Fc protein. TheTables below the diagrams provide the ka, kd and KD values for eachprotein.

FIG. 9 shows the percent inhibition in mice of target induced cytokinesecretion by various amounts of E. coli expressed and refolded, ormammalian expressed, ¹⁰Fn3/Fc protein, indicating that similar levels ofinhibition are obtained with the E. coli expressed and refolded proteinrelative to the mammalian expressed protein. “mpk” refers to milligramsof protein per weight of the animal in kg.

FIG. 10 shows the percent inhibition in mice of target induced cytokine(different from that in FIG. 9) secretion by various amounts of E. coliexpressed and refolded ¹⁰Fn3/Fc protein (“E. coli Refolded”), or¹⁰Fn3/Fc protein expressed in mammalian cells in a shake flask culture(“Mammalian Shake Flask), or bioreactor (“Mammalian Bioreactor”). Alsoshown is the percent inhibition obtained with an antibody (“BMSAntibody”) binding to the same target as one of the targets of the¹⁰Fn3/Fc molecule, as well as inhibition by one of the two ¹⁰Fn3entities on its own (“mono-adnectin”).

FIG. 11 shows the percent inhibition in mice of signal transduction byvarious amounts of E. coli expressed and refolded ¹⁰Fn3/Fc protein (“E.coli Refolded”), or ¹⁰Fn3/Fc protein expressed in mammalian cells in ashake flask culture (“Mammalian Shake Flask), or bioreactor (“MammalianBioreactor”). Also shown is the percent inhibition obtained with anantibody (“BMS Antibody”) binding to the same target as one of thetargets of the ¹⁰Fn3/Fc molecule.

DETAILED DESCRIPTION

Provided herein are methods for refolding denatured proteins, such asproteins present in the form of inclusion bodies (IBs). The methods areapplicable to, e.g., proteins comprising at least one disulfide bond,such as proteins comprising an Fc region or domain (or portions thereof)of antibodies. A method may comprise combining a composition comprisingdenatured or unfolded proteins with a composition having a stronglyalkaline pH, followed by incubation at reduced pH. Unlike commonly usedmethods for refolding proteins, e.g., from IBs, the methods describedherein do not require the use of a denaturing or chaotropic agent. Inaddition, the methods described herein allow refolding of denaturedproteins, e.g., from IBs, without the use of large volumes of buffer andin generally shorter time frames than those of current commonly usedmethods.

Definitions

By “polypeptide” is meant any sequence of two or more amino acids,regardless of length, post-translation modification, or function.Polypeptides can include natural amino acids and non-natural amino acidssuch as those described in U.S. Pat. No. 6,559,126, incorporated hereinby reference. Polypeptides can also be modified in any of a variety ofstandard chemical ways (e.g., an amino acid can be modified with aprotecting group; the carboxy-terminal amino acid can be made into aterminal amide group; the amino-terminal residue can be modified withgroups to, e.g., enhance lipophilicity; or the polypeptide can bechemically glycosylated or otherwise modified to increase stability orin vivo half-life). Polypeptide modifications can include the attachmentof another structure such as a cyclic compound or other molecule to thepolypeptide and can also include polypeptides that contain one or moreamino acids in an altered configuration (i.e., R or S; or, L or D).

A “region” of a ¹⁰Fn3 domain (or moiety) as used herein refers to eithera loop (AB, BC, CD, DE, EF and FG), a β-strand (A, B, C, D, E, F and G),the N-terminus (corresponding to amino acid residues 1-7 of SEQ ID NO:1), or the C-terminus (corresponding to amino acid residues 93-101 ofSEQ ID NO: 1) of the human ¹⁰Fn3 domain having SEQ ID NO: 1.

A “north pole loop” refers to any one of the BC, DE and FG loops of ahuman fibronectin type 3 tenth (¹⁰Fn3) domain.

A “south pole loop” refers to any one of the AB, CD and EF loops of ahuman fibronectin type 3 tenth (¹⁰Fn3) domain.

A “scaffold region” refers to any non-loop region of a human ¹⁰Fn3domain. The scaffold region includes the A, B, C, D, E, F and Gβ-strands as well as the N-terminal region (amino acids corresponding toresidues 1-7 of SEQ ID NO: 1) and the C-terminal region (amino acidscorresponding to residues 93-101 of SEQ ID NO: 1).

“Percent (%) amino acid sequence identity” herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in a selected sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled inthe art can determine appropriate parameters for measuring alignment,including any algorithms needed to achieve maximal alignment over thefull-length of the sequences being compared. For purposes herein,however, % amino acid sequence identity values are obtained as describedbelow by using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., has been filed with user documentation in the U.S. CopyrightOffice, Washington D.C., 20559, where it is registered under U.S.Copyright Registration No. TXU510087, and is publicly available throughGenentech, Inc., South San Francisco, Calif. The ALIGN-2 program shouldbe compiled for use on a UNIX operating system, preferably digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

For purposes herein, the % amino acid sequence identity of a given aminoacid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows: 100times the fraction X/Y where X is the number of amino acid residuesscored as identical matches by the sequence alignment program ALIGN-2 inthat program's alignment of A and B, and where Y is the total number ofamino acid residues in B. It will be appreciated that where the lengthof amino acid sequence A is not equal to the length of amino acidsequence B, the % amino acid sequence identity of A to B will not equalthe % amino acid sequence identity of B to A.

As used herein, an amino acid residue in a polypeptide is considered to“contribute to binding” a target if (1) any of the non-hydrogen atoms ofthe residue's side chain or main chain is found to be within fiveangstroms of any atom of the binding target based on an experimentallydetermined three-dimensional structure of the complex, and/or (2)mutation of the residue to its equivalent in wild-type ¹⁰Fn3 (e.g., SEQID NO: 1), to alanine, or to a residue having a similarly sized orsmaller side chain than the residue in question, leads to a measuredincrease of the equilibrium dissociation constant to the target (e.g.,an increase in the k_(on)).

“Moiety” refers to a portion of a protein. For example, a fusion proteinmay comprise several moieties. In one embodiment, a fusion proteincomprises a fibronectin based scaffold moiety and an Fc moiety. An Fcmoiety may comprise a CH2 and a CH3 domain, but does not necessarilycomprise a hinge.

A “denatured protein” refers to a protein that is not properly folded(i.e., does not have the proper spatial conformation or threedimensional structure). “Denaturation” refers to a process in which thenative conformation of the protein is changed but the primary structure(amino acid chain, peptide links) of the protein remains unchanged. Tobe able to perform its biological function, a protein folds into aspecific spatial conformation, by the action of non-covalentinteractions such as ionic interactions, Van Der Waals forces, hydrogenbonding, and hydrophobic packing A protein that is denatured (i.e., notproperly folded) may be a protein that does not have a proper secondary,tertiary or quaternary structure. The secondary structure of a proteinor polypeptide refers to highly regular local sub-structures, such asthe alpha helix and the beta strand or beta sheets, of a protein. Thetertiary structure of a protein or a polypeptide refers to thethree-dimensional structure of a single protein molecule, in which thefolding of the alpha-helices and beta-sheets into a compact globule isdriven by the non-specific hydrophobic interactions (the burial ofhydrophobic residues from water), salt bridges, hydrogen bonds, and thetight packing of side chains and disulfide bonds. The quaternarystructure of a protein is the three-dimensional structure of subunits ofa multi-subunit protein. The subunits of a protein are held together bythe same bonds as those that maintain a tertiary structure of a protein.Disulfide bonds contribute to the tertiary and quaternary structure of aprotein, polypeptide or polypeptide complex. A denatured protein may bea protein that contains cysteines, but in which the disulfide bonds arenot present or are improperly formed. Denatured proteins are generallyinsoluble and precipitate out of a solution. The presence of one or moredisulfide bonds in a protein generally makes its renaturation from adenatured state more challenging. Protein structure can be visualized ordetermined with various tools, e.g., X-ray crystallography, NuclearMagnetic Resonance (NMR), circular dichroism and cryo-electronmicroscopy.

Methods for Refolding Denatured Proteins

When proteins are expressed in certain expression systems, they areproduced in a denatured form and must be renatured, i.e., theirsecondary, tertiary and/or quaternary structure must be reformed. Forexample, proteins expressed at high levels in E. coli are shunted intoinclusion bodies (IBs). IBs are essentially made of denatured proteins.The methods described herein may be used to renature unfolded orimproperly folded proteins, e.g., present in IBs.

A method for refolding a denatured protein, may comprise: (i) combiningdenatured protein with a solubilization buffer having a pH in the rangeof 10 to 13 to thereby obtain a composition comprising solubilizeddenatured protein; and (ii) combining the composition comprisingsolubilized denatured protein, with a refold buffer having a pH in therange of 9 to 11 to thereby obtain a composition comprising refoldedprotein. A method for refolding a denatured protein, may comprise: (i)suspending the denatured protein in a suspension solution to obtain acomposition comprising suspended denatured protein; (ii) combining thecomposition comprising suspended denatured protein with a solubilizationbuffer having a pH in the range of 10 to 13 to thereby obtain acomposition comprising solubilized denatured protein; and (iii)combining the composition comprising solubilized denatured protein, witha refold buffer having a pH in the range of 9 to 11 to thereby obtain acomposition comprising refolded protein. When a denatured protein ispart of an IB, a method for refolding the protein may comprise: (i)combining the IBs with a solubilization buffer having a pH in the rangeof 10 to 13 to thereby obtain a composition comprising solubilized IBs;and (ii) combining the composition comprising solubilized IBs with arefold buffer having a pH in the range of 9 to 11 to thereby obtain acomposition comprising refolded protein. A method for refolding aprotein may comprise (i) suspending IBs comprising a protein in an IBsuspension solution to obtain a composition comprising suspended IBs;(ii) combining the composition comprising suspended IBs with asolubilization buffer having a pH in the range of 10 to 13 to therebyobtain a composition comprising solubilized IBs; and (iii) combining thecomposition comprising solubilized IBs with a refold buffer having a pHin the range of 9 to 11 to thereby obtain a composition comprisingrefolded protein. A diagram showing exemplary refolding steps isprovided in FIG. 1. The first step of the method may be removed.

Certain commonly used methods for refolding denatured proteins known inthe art use denaturing agents for solubilizing the denatured proteins.In certain embodiments, the methods described herein or one or more ofthe compositions, buffers or solutions used in the methods describedherein, do not include a significant amount of a denaturing agent.Exemplary denaturing (or chaotropic) agents include: guanidine (orguanidium), guanidium hydrochloride, guanidium chloride, guanidiumthiocyanate, urea, thiourea, lithium perchlorate, magnesium chloride,phenol, betain, sarcosine, carbamoyl sarcosine, taurine,dimethylsulfoxide (DMSO); alcohols such as propanol, butanol andethanol; detergents, such as sodium dodecyl sulfate (SDS), N-lauroylsarcosine, Zwittergents, non-detergent sulfobetains (NDSB), TRITON™X-100, NONIDET™ P-40, the TWEEN™ series and BRIJ™ series; hydroxidessuch as sodium potassium hydroxide, and combinations thereof. A“significant” amount of a denaturant refers to an amount of denaturantthat is sufficient to contribute to the solubilization of denaturedprotein. Certain concentrations of denaturants denature proteins, andthese concentrations are referred to as “denaturing concentrations.”Certain concentrations of denaturants do not denature proteins, but maycontribute to the solubilization of proteins, and these concentrationsare referred to as “non-denaturing concentrations.” For example, 6 Mguanidium is a denaturing concentration, whereas 1M guanidium is not adenaturing concentration. Similarly, urea concentrations of 1-2 M arenot considered to be denaturing concentrations. A solution comprisingless than a significant amount of a denaturant includes solutionscomprising less than a denaturing or non-denaturing concentration of adenaturant. For example, the methods described herein preferably do notinclude urea or guanidium at a concentration of 1M or more. In certainembodiments, minor amounts of any denaturing agent that is low enoughthat it does not contribute to denaturation and/or solubilization of adenatured protein may be present at any time in the methods describedherein, e.g., in one or more of the compositions, buffers and solutionsused in the methods described herein. As defined herein, when referringto methods which do not include the use of a denaturing agent or asignificant amount of a denaturing agent, the agent that produces thealkaline conditions in the solubilization buffer is not considered to bea denaturing agent, or in the alternative, if it is considered to be adenaturing agent, then the statement is intended to mean that no otherdenaturing agent is added or used.

In certain embodiments, a minor amount of a denaturant is an amount ofdenaturant that is sufficiently low that its inclusion in a refoldingmethod does not require an additional step to later reduce itsconcentration or remove it from a solution. For example, in certainembodiments, the methods described herein do not include a dialysisstep, e.g., they do not include a dialysis step before or after any ofthe steps of the methods described herein. For example, no dialysisprior to, or after, adding the suspension solution, solubilizationbuffer or refold buffer is performed.

In certain embodiments, the concentration of a denaturing agent in themethods described herein or in any step of the methods described hereinis less than 1M, 100 mM 10 mM, 1 mM, 0.1 mM, 10⁻² mM, 10⁻³ mM, 10⁻⁴ mM,10⁻⁵ mM, or 10⁻⁶ mM. In certain embodiments, no denaturing agent isadded to, or present in, one or more of the following solutions used inthe methods described herein: the suspension solution, such as an IBsuspension solution; the solubilization buffer; and the refold buffer.In certain embodiments, no denaturing agent is used or present in anystep in the methods described herein.

In certain embodiments, the concentration of guanidium or salt or analogthereof (e.g., guanidium chloride, guanidium hydrochloride and guanidiumthiocyanate) in any step of the methods described herein is less than1M, 100 mM, 10 mM, 1 mM, 0.1 mM, 10⁻² mM, 10⁻³ mM, 10⁻⁴ mM, 10⁻⁵ mM, or10⁻⁶ mM. In certain embodiments, no guanidium, salt or analog thereof(e.g., guanidium chloride, guanidium hydrochloride and guanidiumthiocyanate) is added to, or present in, one or more of the followingsolutions used in the methods described herein: the suspension solution,such as an IB suspension solution; the solubilization buffer; and therefold buffer. In certain embodiments, no guanidium, salt or analogthereof (e.g., guanidium chloride, guanidium hydrochloride and guanidiumthiocyanate) is used or present in any step in the methods describedherein. In certain embodiments, the concentration of urea or analogthereof (e.g., dimethylhydroxy urea, dimethylsulphone) in any step ofthe methods described herein is less than 1M, 100 mM, 10 mM, 1 mM, 0.1mM, 10⁻² mM, 10⁻³ mM, 10⁻⁴ mM, 10⁻⁵ mM, or 10⁻⁶ mM. In certainembodiments, no urea or analogs thereof (e.g., dimethylhydroxy urea anddimethylsulphone) is added to, or present in, one or more of thefollowing solutions used in the methods described herein: the suspensionsolution, such as an IB suspension solution; the solubilization buffer;and the refold buffer. In certain embodiments, no urea or analogsthereof (e.g., dimethylhydroxy urea and dimethylsulphone) is used orpresent in any step in the methods described herein. In certainembodiments, the concentration of a detergent, e.g., ionic or non-ionic,in any step of the methods described herein is less than 1M, 100 mM, 10mM, 1 mM, 0.1 mM, 10⁻² mM, 10⁻³ mM, 10⁻⁴ mM, 10⁻⁵ mM, or 10⁻⁶ mM or lessthan 10%, 1%, 0.1%, 0.01% or 0.001% final concentration. In certainembodiments, no detergent, e.g., ionic or non-ionic, is added to, orpresent in, one or more of the following solutions used in the methodsdescribed herein: the suspension solution, such as an IB suspensionsolution; the solubilization buffer; and the refold buffer. In certainembodiments, no detergent, e.g., ionic or non-ionic, is used or presentin any step in the methods described herein. Non-ionic detergentsinclude TRITON™ X-100, NONIDET™ P-40, the TWEEN™ series and BRIJ™series. Ionic detergents include deoxycholate, SDS, and CTAB.

In certain embodiments, the methods described herein do not use asignificant amount of a reducing agent. A reducing agent is an agentthat breaks disulfide bonds by reducing one or the two cysteines of thedisulfide bond or maintains cysteines in a reduced state (i.e.,maintains free sulfhydryl groups so that the intra- or intermoleculardisulfide bonds are chemically disrupted). A “significant amount” of areducing agent is an amount that is sufficient for reducing at leastsome disulfide bonds in a protein solution or for maintaining at leastsome cysteines in a protein solution in a reduced state. Exemplaryreducing agents include the following: beta-mercaptoethanol (BME),dithiothreitol (DTT), dithioerythritol (DTE),tris(2-carboxyethyl)phosphine (TCEP), cysteine, cysteamine,thioglycolate, glutathione and sodium borohydride. In certainembodiments, no reducing agent is added to, or present in, one or moreof the following solutions used in the methods described herein: thesuspension solution, such as an IB suspension solution; thesolubilization buffer; and the refold buffer. In certain embodiments, noreducing agent is used or present in any step in the methods describedherein. In certain embodiments, the concentration of a reducing agent inany step of the methods described herein is less than 10 mM, 1 mM, 0.1mM, 10⁻² mM, 10⁻³ mM, 10⁻⁴ mM, 10⁻⁵ mM, or 10⁻⁶ mM.

A method for refolding a denatured protein, e.g., a protein that ispresent in IBs, may include washing the denatured protein, e.g., IBs,prior to suspending them in a suspension solution. Washing denaturedprotein, e.g., IBs, may be performed with, e.g., Tris/HCL buffer,phosphate buffer, acetate buffer, citrate buffer or water, or acombination of two or more of these.

Suspension of Denatured Protein

Denatured protein, e.g., protein in IBs, which denatured protein may be,e.g., in the form of a pellet (such as a frozen pellet) may be suspendedin a suspension solution (or buffer). The denatured protein may beincubated with suspension solution under conditions sufficient tosubstantially suspend the denatured protein. Incubation may take placeunder conditions of concentration, incubation time, and incubationtemperature to allow suspension of the desired amount or most orsubstantially all the denatured protein (e.g., at least 70%, 80%, 90%,95%, 97%, 98% or 99%).

In certain embodiments, the suspension solution is water. Water may be,e.g., tap water, distilled, double distilled, deionized water, reverseosmosed water, or reversed osmosed/deionized (RODI) water. In certainembodiments, a suspension solution comprises low concentrations of abuffer, e.g., TRIS/HCL, e.g., less than about 10 mM, 1 mM, 0.1 mM orless TRIS. A suspension solution may have a pH of 6-10, 6-9, 6-8, 6.5 to7.5.

In certain embodiments, a pellet of denatured protein is contacted witha suspension solution at a ratio of weight of denatured protein pellet(e.g., IB) (in grams):volume (in ml) of suspension solution (e.g., IBsuspension solution) of 1:1-10; 1:1-9; 1:1-8; 1:1-7; 1:1-6; 1:1-5;1:1-4; 1:1-3; 1:1-2; 1:1; 1:2; 1:3; 1:4; 1; 5; 1:6; 1:7; 1:8 1:9 or1:10. In certain embodiments, a pellet of denatured protein is contactedwith a suspension solution at a ratio of weight of denatured proteinpellet (e.g., IB) (in grams):volume (in ml) of suspension solution(e.g., IB suspension solution) of 1:1-3. A weight to volume ratio of“1:3” in this context refers to a ratio of 1 gram of denatured proteinto 3 ml of suspension solution. A weight to volume ratio of “1:1-3” inthis context refers to a ratio of 1 gram of denatured protein to 1-3 ml(e.g., 1 ml, 2 ml or 3 ml and any values in between) of suspensionsolution. A weight to volume ratio may also be defined in kgs:liters.The combination of the denatured proteins and the suspension solution isreferred to as the “suspension reaction.”

The suspension reaction may be conducted at a temperature, e.g., rangingfrom 2° C. to 40° C.; 4° C. to 37° C.; 25° C. to 37° C.; roomtemperature; or 4° C. to 25° C. In an exemplary embodiment, a pellet ofdenatured protein, e.g., an IB pellet, is suspended in water at roomtemperature (e.g., 25° C.) at a weight (grams) to volume (ml) ratio ofdenatured protein pellet:suspension solution of 1:1-3, such as 1:1, 1:2or 1:3.

A suspension reaction may be incubated, and optionally stirred, with asuspension solution until most or essentially all denatured protein hasbeen resuspended, and optionally a fine suspension is obtained. Anyportion of a pellet of denatured protein that has not fully beensuspended will probably not be renatured efficiently. The proportion ofdenatured protein that is suspended in the suspension solution may bedetermined optically. In certain embodiments, the denatured protein isincubated and optionally stirred, e.g., for less than 1 minute, in thesuspension buffer. In certain embodiments, the denatured protein isincubated and optionally stirred, e.g., for 1-10 minutes; 1-5 minutes or1-3 minutes in the suspension solution. Longer incubation times,especially at lower temperatures may also be used.

In certain embodiments, a pellet of denatured protein is suspended inwater at a weight (grams) to volume (ml) ratio of denatured proteinpellet:volume of suspension solution of 1:1-3, e.g., 1:2, at roomtemperature and incubated at room temperature for 1-3 minutes, tothereby obtain a composition comprising a suspension of denaturedproteins. In an exemplary embodiment, a pellet of IBs is suspended inwater at a weight (grams) to volume (ml) ratio of IB:suspension solutionof 1:1-3, e.g., 1:2, at room temperature and incubated at roomtemperature for 1-3 minutes, to thereby obtain a composition comprisingan IB suspension.

In certain embodiments, the suspension solution or suspension reactiondoes not comprise a significant amount of denaturing agent, as furtherdescribed herein. In certain embodiments, the suspension solution orsuspension reaction does not comprise a significant amount of reducingagent, as further described herein. In certain embodiments, thesuspension solution or suspension reaction comprises neither asignificant amount of denaturing agent nor a significant amount ofreducing agent.

Solubilization of Denatured Protein

A suspension of denatured proteins, e.g., an IB suspension, (obtained,e.g., as described above) may be combined with a solubilization bufferto thereby obtain a composition comprising solubilized denaturedprotein, e.g., solubilized IBs. In certain embodiments, a the denaturedprotein, e.g., in the form of a pellet, is directly combined withsolubilization buffer, without prior suspension. The denatured protein,e.g., in the form of a suspension of denatured proteins, may beincubated with solubilization buffer under conditions sufficient tosubstantially solubilize the protein. Incubation may take place underconditions of concentration, incubation time, and incubation temperatureto allow solubilization of the desired amount or most or substantiallyall the protein (e.g., at least 70%, 80%, 90%, 95%, 97%, 98% or 99%).

In certain embodiments, a solubilization buffer comprises a bufferingagent suitable for maintaining the pH of the solubilization bufferand/or that of the composition comprising the solubilization buffer anddenatured protein (“solubilization reaction”) in a range of pH 10 to 13.The pH of the solubilization buffer and/or the solubilization reactionmay also be within the following ranges of pH: pH 10.5 to 13; pH 11 to13; pH 11 to 12.8; pH 11.5 to 12.8; pH 11.8 to 12.6; pH 12.0 to 12.6; pH12.0 to 12.4 and pH 12.2 to 12.5. Exemplary pHs of solubilizationbuffers and/or solubilization reactions include pH 12.0; pH 12.1; pH12.2; pH 12.3; pH 12.4 and pH 12.5.

A solubilization buffer may comprise Arginine (or another positivelycharged amino acid), e.g., L-arginine/HCl (which is encompassed by theterm “Arginine”). A solubilization buffer may comprise Arginine at aconcentration that is sufficient for buffering the solubilization bufferat the desired pH, e.g., a pH in the range of pH 10.5 to 13; such as pH12.0 to pH 12.5. Arginine may be present at concentrations in the rangeof 50 mM to 500 mM; 100 mM to 500 mM; 200 mM to 500 mM; 300 mM to 500mM; 350 mM to 450 mM. In certain embodiments, the solubilization bufferincludes Arginine at a concentration of 300 mM to 400 mM. In certainembodiments, a solubilization buffer comprises Arginine at 50 mM to 500mM and has a pH in the range of 10.5 to 13. In certain embodiments, asolubilization buffer comprises Arginine at 200 mM to 500 mM and has apH in the range of 12 to 12.4.

As Arginine buffers the pH of a solution to an alkaline value, it is notnecessary to include another buffer in the solubilization buffer.However, in certain embodiments, one may include one or more of thefollowing buffers: TRIS (Tris[hydroxymethyl]aminomethane), HEPES(N-[2-Hydroxyethyl]piperazine-N′-[3-propane-sulfonic acid]), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP(2-Amino-2-methyl-1-propanol), CAPS(3-[Cyclohexylamino]-1-propanesulfonic acid), CHES(2-[N-Cyclohexylamino]ethanesulfonic acid), arginine, lysine, and sodiumborate. In certain embodiments, the solubilization buffer comprises abuffer, e.g., TRIS, at a concentration of 1 mM to 1 M; 1 mM to 100 mM;10 mM to 100 nM; 10 mM to 50 mM; 50 mM to 100 mM; 30 mM to 70 mM; or 40mM to 60 mM. In certain embodiments, the solubilization buffer comprisesa buffer, e.g., TRIS, at a concentration of about 50 mM. In certainembodiments, the solubilization buffer comprises TRIS, e.g., at 40-60mM, and has a pH in the range of pH 12.0 to 12.4.

The solubilization buffer may comprise TRIS and Arginine and have a pHin the range of 12.0 to 12.4. In certain embodiments, the solubilizationbuffer comprises TRIS at a concentration in the range of 10 mM to 100mM; Arginine; and have a pH in the range of pH 12.0 to 12.4. Thesolubilization buffer may comprise TRIS and Arginine, wherein Arginineis at a concentration in the range of 300 mM to 500 mM; and has a pH inthe range of 12.0 to 12.4. The solubilization buffer may comprise TRISat a concentration in the range of 10 mM to 100 mM; Arginine at aconcentration in the range of 300 mM to 500 mM; and have a pH in therange of pH 12.0 to 12.4. In certain embodiments, the solubilizationbuffer comprises TRIS at a concentration in the range of 30 mM to 70 mM;Arginine at a concentration in the range of 300 mM to 500 mM; and has apH in the range of pH 12.0 to 12.4. In certain embodiments, thesolubilization buffer comprises TRIS at a concentration of about 50 mM,Arginine at a concentration of about 400 mM, and has a pH of about 12.2.

The composition comprising the suspended denatured protein, e.g.,suspended IBs, may be combined with solubilization buffer at a weight(in grams) to volume (in ml) ratio of weight of denatured protein (e.g.,IBs):volume of solubilization buffer of 1:5-50; 1:10-50; 1:10-30;1:15-25. Exemplary ratios include 1:10, 1:20 and 1:30. For example, for1 gram of denatured protein (that was suspended in suspension solution),5-50 ml; 10-50 ml; 10 to 30 ml or 15 to 25 ml of solubilization buffermay be added. In other embodiments, for 1 kg of denatured protein (thatwas suspended in suspension solution), 5-50 liters; 10-50 liters; 10 to30 liters or 15 to 25 liters of solubilization buffer may be added.

The solubilization reaction may be conducted at a temperature, e.g.,ranging from 2° C. to 40° C.; 4° C. to 37° C.; 25° C. to 37° C.; roomtemperature; or 4° C. to 25° C. In certain embodiments, thesolubilization reaction is conducted at room temperature (e.g., 25° C.)at a weight (in grams) to volume (in ml) ratio of weight of denaturedprotein (e.g., IBs):volume of solubilization buffer of 1:10-30, e.g.,1:10, 1:20 or 1:30.

The composition comprising the suspended denatured proteins and thesolubilization buffer (the “solubilization reaction”) is incubated, andoptionally stirred, for a time sufficient to solubilize essentially all(e.g., at least 70%, 80%, 90%, 95%, 97%, 98% or 99%) of the protein,e.g., prior to adding the refold buffer. The solubilization reaction maybe incubated for less than 1 minute; 1 minute to 6 hours; 1 minute to 5hours; 1 minute to 3 hours; 1 minute to 2 hours; 1 to 60 minutes; 1 to30 minutes; 1 to 20 minutes; 1 to 10 minutes; 1 to 5 minutes; 1 to 3minutes; 1 to 2 minutes; 2 to 5 minutes or 2 to 3 minutes, e.g., priorto adding the refold buffer. The solubilization reaction is preferablyperformed for a time frame sufficient for most proteins to besolubilized. That most or essentially all of the proteins have beensolubilized in the solubilization buffer can be determined optically, asthe solution becomes clear (transparent) once all or most of theproteins have been solubilized. At the same time, it is preferable tokeep the incubation time of the solubilization reaction as short aspossible as deamidation occurs at high pH values. For example,incubation of the solubilization reaction may be conducted for a timethat results in less than 15%; 12%; 10%; 7%; 5%; 3%; 2% or 1%deamidation (corresponding, e.g., to total additive percentage ofdeamidation and isoaspartate formation at multiple sites). Deamidationcan be measured, e.g., by Liquid Chomatography Mass Spectrometry(LCMS)/peptide map analysis.

In certain embodiments, a suspension of denatured proteins, e.g., IBs,is combined, and optionally mixed, with a solubilization buffercomprising Arginine at a concentration in the range of 300 mM to 500 mMand optionally TRIS at a concentration in the range of 30 mM to 70 mM;and having a pH in the range of 12.0 to 12.4 at a ratio of weight(grams) of denatured protein:volume (ml) of solubilization buffer of1:10-30, at room temperature; and wherein incubation is conducted for 2to 5 minutes prior to, e.g., the addition of refold buffer, to therebyobtain a composition comprising solubilized denatured proteins, e.g.,solubilized IBs.

In certain embodiments, the solubilization buffer or solubilizationreaction does not comprise a significant amount of denaturing agent, asfurther described herein. In certain embodiments, the solubilizationbuffer or solubilization reaction does not comprise a significant amountof reducing agent, as further described herein. In certain embodiments,the solubilization buffer or solubilization reaction comprises neither asignificant amount of denaturing agent nor a significant amount ofreducing agent.

Protein concentration during the solubilization step may be about 1mg/ml to about 60 mg/ml, e.g., about 10-50 mg/ml.

Refolding of Denatured Protein

A composition comprising solubilized denatured protein, e.g., acomposition comprising solubilized IBs, (obtained, e.g., as describedabove) may be combined with a refold buffer to thereby obtain acomposition comprising refolded protein. The composition comprisingsolubilized denatured proteins may be incubated with refold buffer underconditions sufficient to substantially refold the protein. Incubationmay take place under conditions of concentration, incubation time, andincubation temperature to allow refolding of the desired amount or mostor substantially all the protein (e.g., at least 70%, 80%, 90%, 95%,97%, 98% or 99%).

In certain embodiments, a refold buffer comprises a buffering agentsuitable for maintaining the pH of the refold buffer and/or that of thecomposition comprising the solubilized refold buffer and solubilizedprotein (the “refold reaction”) in a range of pH of 9 to 11. The pH ofthe refold buffer and/or the refold reaction may also be within thefollowing ranges of pH: pH 9 to 11; pH 9.5 to 11; pH 10 to 11; pH 10 to10.5; pH 10 to 10.8; and pH 10.2 to 10.6. Exemplary pHs of refoldbuffers and/or refold reactions include pH 10.0; pH 10.1; pH 10.2; pH10.3; pH 10.4; pH 10.5; pH 10.6 and pH 10.7.

Refold buffer may comprise Arginine (or another positively charged aminoacid), e.g., L-arginine/HCl (which is encompassed by the term“Arginine”). A refold buffer may comprise Arginine at a concentrationthat is sufficient for buffering the solubilization buffer at thedesired pH, e.g., a pH in the range of pH 9 to 11; such as pH 10 to10.8. Arginine may be present at concentrations in the range of 50 mM to500 mM; 100 mM to 500 mM; 200 mM to 500 mM; 300 mM to 500 mM; 350 mM to450 mM. In certain embodiments, the refold buffer includes Arginine at aconcentration of 300 mM to 400 mM. In certain embodiments, a refoldbuffer comprises Arginine at 50 mM to 500 mM and has a pH in the rangeof 9 to 11. In certain embodiments, a refold buffer comprises Arginineat 200 mM to 500 mM and has a pH in the range of pH 10 to 10.8.

As Arginine is buffering the refold solution at pH 10.4, it is notnecessary to include another buffer. However, if desired, any of thefollowing buffers may be added: TRIS (Tris[hydroxymethyl]aminomethane),HEPPS (N-[2-Hydroxyethyl]piperazine-N′-[3-propane-sulfonic acid]), CAPSO(3-[Cyclohexylamino]-2-hydroxy-1-propanesulfonic acid), AMP(2-Amino-2-methyl-1-propanol), CAPS(3-[Cyclohexylamino]-1-propanesulfonic acid), CHES(2-[N-Cyclohexylamino]ethanesulfonic acid), arginine, lysine, and sodiumborate. In certain embodiments, the refold buffer includes a buffer,e.g., TRIS, at a concentration of 1 mM to 1 M; 1 mM to 100 mM; 10 mM to100 nM; 10 mM to 50 mM; 50 mM to 100 mM; 30 mM to 70 mM; or 40 mM to 60mM. In certain embodiments, the refold buffer comprises a buffer, e.g.,TRIS, at a concentration of about 50 mM. In certain embodiments, therefold buffer comprises TRIS, e.g., at 40-60 mM, and has a pH in therange of pH 10.2 to 10.6.

The refold buffer may comprise TRIS and Arginine and have a pH in therange of pH 10.2 to 10.6. In certain embodiments, the refold buffercomprises TRIS at a concentration in the range of 10 mM to 100 mM;Arginine; and has a pH in the range of pH 10.2 to 10.6. The refoldbuffer may comprise TRIS and Arginine, wherein Arginine is at aconcentration in the range of 300 mM to 500 mM; and have a pH in therange of pH 10.2 to 10.6. The refold buffer may comprise TRIS at aconcentration in the range of 10 mM to 100 mM; Arginine at aconcentration in the range of 300 mM to 500 mM; and have a pH in therange of pH 10.2 to 10.6. In certain embodiments, the refold buffercomprises TRIS at a concentration in the range of 30 mM to 70 mM;Arginine at a concentration in the range of 300 mM to 500 mM; and have apH in the range of pH 10.2 to 10.6. In certain embodiments, thesolubilization buffer comprises TRIS at a concentration of about 50 mM,Arginine at a concentration of about 400 mM, and has a pH of about 10.4.

In embodiments, in which the protein to be refolded comprise one or moredisulfide bonds when properly folded, the refold buffer may alsocomprise an oxidizing agent to facilitate the formation of disulfidebonds. For refolding proteins that do not comprise a disulfide bond, itis not necessary to include an oxidizing agent. In certain embodiments,the oxidizing agent comprises glutathione, e.g., in a ratio of oxidizedglutathione:reduced glutathione of about 5:1 or a similar ratiosufficient to facilitate the formation of disulfide bonds. In certainembodiments, a refold buffer comprises 0.1 mM to 10 mM of oxidizedglutathione and 0.02 mM to 2 mM of reduced glutathione. In certainembodiments, a refold buffer comprises 0.5 mM to 2 mM of oxidizedglutathione and 0.1 to 0.4 mM of reduced glutathione. In certainembodiments, a refold buffer comprises about 1 mM of oxidizedglutathione and about 0.2 mM of reduced glutathione. Other oxidizingagents known in the art may also be used.

A composition comprising solubilized protein may be combined with refoldbuffer at a ratio of volume (ml) of solubilization buffer used tosolubilize the denatured protein:volume (ml) of refold buffer of 1:1-50;1:1-20; 1:1-10; 1:1-5; 1:2-10; 1:2-8; or 1:2-5. Exemplary ratios includeabout 1:1, 1:2, 1:3, 1:4; 1:5 or 1:6.

The refold reaction may be conducted at a temperature, e.g., rangingfrom 2° C. to 40° C.; 4° C. to 37° C.; 25° C. to 37° C.; roomtemperature; or 4° C. to 25° C. In certain embodiments, the refoldreaction is conducted at room temperature (e.g., 25° C.) at a ratio ofvolume (ml) of solubilization buffer used to solubilize the denaturedprotein:volume (ml) of refold buffer of 1:1-5, e.g., 1:1, 1:3 or 1:5.

Refold occurs essentially instantaneously, and is generally performedfor a time frame sufficient for most proteins to be refolded. In certainembodiments, the refold reaction may be incubated (e.g., with or withoutstirring), e.g., overnight; for 1 minute to 12 hours; 1 minute to 6hours; 1 minute to 3 hours; 1 to 120 minutes; 1 to 30 minutes; 1 to 20minutes; 1 to 10 minutes; 1 to 100 minutes; 10 to 100 minutes; 10 to 80minutes; 20 to 60 minutes prior to, e.g., adjusting the pH down.Incubation may be performed with or without stirring. In certainembodiments, the refold reaction is stirred and then incubated withoutstirring.

In certain embodiments, a composition comprising solubilized proteins iscombined with a refold buffer comprising Arginine at a concentration inthe range of 300 mM to 500 mM; oxidized glutathione at a concentrationin the range of 0.5 mM to 2 mM; (optionally TRIS at a concentration inthe range of 30 mM to 70 mM) reduced glutathione at a concentration inthe range of 0.1 to 0.4 mM, and having a pH in the range of 10.2 to10.6, at a volume ratio of solubilization buffer:refold buffer of 1:1-5,and incubated at room temperature for 1 minute to overnight, prior to,e.g., adjusting the pH to a lower value.

In certain embodiments, the refold buffer or refold reaction does notcomprise a significant amount of denaturing agent, as further describedherein. In certain embodiments, the refold buffer or refold reactiondoes not comprise a significant amount of reducing agent (except whenco-administered together with an oxidant, e.g., when oxidized andreduced glutathione are added together), as further described herein. Incertain embodiments, the refold buffer or refold reaction comprisesneither a significant amount of denaturing agent nor a significantamount of reducing agent.

Following the refolding reaction, the pH may be adjusted to a lowervalue, e.g., pH 6 to 8 or pH 7 to 8. In certain embodiments, the pH isadjusted to about pH 8. In certain embodiments, adjusting the pH down toabout pH 8 comprises adding 0.3 fold volume of 1M HCl. The addition ofthe HCl may be conducted slowly, e.g., over 0.5 to 2 minutes.

Following the adjustment to a lower pH, the reaction mixture may beincubated for 30 minutes to 3 hours; for 30 minutes to 2 hours, orovernight, prior to a next step, e.g., a purification step. A refoldedprotein may be further processed, e.g., purified, according to methodsknown in the art, e.g., using protein A chromatography and other typesof chromatography or purification methods.

Protein concentration during the refold step may be from 1 mg/ml or lessto about 10 mg/ml. For example, the protein concentration may be about 5mg/ml, 6 mg/ml or 7 mg/ml.

In certain embodiments, total recovery of protein refolded as describedherein may be greater than 70% or 80% as measured by reverse phasechromatography. Total downstream processing recoveries may be as high as20%, 30%, 40% or more from solubilization of the denatured protein,e.g., IBs, through final chromatography.

During standard expression and purification of recombinant proteincomprising an Fc, the disulfide bond of the CH3 loop breaks in about0.5-5% of the protein composition. Using certain methods describedherein, the CH3 open loop has been shown to be in the range of 0.5-3% inthe refolded proteins. Accordingly certain Fc containing proteincompositions, wherein the proteins have been refolded as describedherein, have less than 5%, 4%, 3%, 2%, 1% CH3 open loops. It has alsobeen shown herein that deamidation can be reduced from 40% to 12% (totaladditive percentage of deamidation and isoasp formation at multiplesites). Accordingly, in certain embodiments, protein refolded using amethod described herein comprise less than 40%, 30%, 20%, 15%, or 13%deamidation.

In certain embodiments, refolding denatured protein, e.g., from an IB,is performed in less than 4 hours, 3 hours, or 2 hours.

Exemplary Methods

A method for refolding a denatured protein, e.g., from an IB, maycomprise: (i) suspending a pellet of denatured protein, e.g., an IBpellet, in suspension solution (e.g., water) at a ratio of weight(grams) of denatured protein pellet:volume (ml) of suspension solutionof 1:1-3 at room temperature for a time sufficient for most of thedenatured protein to be suspended, e.g., 1-10 minutes, to thereby obtaina composition comprising a suspension of denatured protein; (ii)combining (and optionally mixing) the composition comprising asuspension of denatured protein with a solubilization buffer comprisingArginine at a concentration sufficient to buffer the solubilizationbuffer to a pH in the range of pH 12.0 to 12.4, e.g., in the range of100 mM to 500 mM (and optionally TRIS at a concentration in the range of10 mM to 100 mM); at a ratio of weight (grams) of denaturedprotein:volume (ml) of solubilization buffer of 1:10-30, wherein theincubation is conducted at room temperature for a time sufficient tosolubilize most of the denatured protein, e.g., 2 to 5 minutes, tothereby obtain a composition comprising solubilized denatured protein;and (iii) combining the composition comprising solubilized denaturedprotein with a refold buffer comprising Arginine at a concentrationsufficient to buffer the refold buffer to a pH in the range of pH 10.2to 10.6, e.g., at a concentration in the range of 100 mM to 500 mM (andoptionally TRIS at a concentration in the range of 10 mM to 100 mM); aconcentration of oxidizing agent sufficient to promote disulfide bondformation, e.g., oxidized glutathione at a concentration in the range of0.5 mM to 2 mM and reduced glutathione at a concentration in the rangeof 0.1 mM to 0.4 mM; at a ratio of volume of solubilization buffer usedin step (ii):volume of refold buffer of 1:1-5, wherein the incubation isconducted at room temperature for a time sufficient to refold most ofthe protein, e.g., for 1 minute to overnight; and wherein the methoddoes not comprise using a significant amount of denaturing agent andoptionally does not comprise using a significant amount of a reducingagent (other than the reduced agent that is used together with theoxidizing agent).

In certain embodiments, a method for refolding a denatured protein,e.g., from an IB, comprises: (i) suspending a pellet of denaturedprotein, e.g., an IB pellet, in water at a volume ratio of weight(grams) of denatured protein pellet:volume (ml) of water of 1:1-3 atroom temperature for 1-10 minutes to thereby obtain a compositioncomprising a suspension of denatured protein; (ii) combining and mixingthe composition comprising a suspension of denatured protein with asolubilization buffer comprising Arginine at a concentration in therange of 300 mM to 500 mM (and optionally TRIS at a concentration in therange of 30 mM to 70 mM); and having a pH in the range of pH 12.0 to12.4; at a ratio of weight (grams) of denatured protein:volume (ml) ofsolubilization buffer of 1:10-30, wherein the incubation is conducted atroom temperature for 2 to 5 minutes, to thereby obtain a compositioncomprising solubilized denatured protein; and (iii) combining thecomposition comprising solubilized denatured protein with a refoldbuffer comprising Arginine at a concentration in the range of 300 mM to500 mM (and optionally TRIS at a concentration in the range of 30 mM to70 mM); oxidized glutathione at a concentration in the range of 0.5 mMto 2 mM; reduced glutathione at a concentration in the range of 0.1 mMto 0.4 mM; and having a pH in the range of pH 10.2 to 10.6; at a ratioof volume of solubilization buffer used in step (ii):volume of refoldbuffer of 1:1-5, wherein the incubation is conducted at room temperaturefor 1 to 120 minutes; and wherein the method does not comprise using asignificant amount of denaturing agent and optionally does notcomprising using a significant amount of a reducing agent (other thanthe reduced agent that is used together with the oxidizing agent).

Generally, the margin of error of pH for a prepared buffer solution is+/−0.1 units.

Exemplary Proteins

Proteins that may be refolded from a denatured state, e.g., from IBs,using the methods described herein include any protein that is in adenatured form, e.g., proteins comprising at least one disulfide bond intheir native state. Proteins without disulfide bonds may also berefolded as described herein. Proteins may comprise a binding domainthat specifically binds to a target protein. A protein may be anaturally occurring protein or a genetically engineered or fusionprotein. An exemplary protein that may be refolded as described hereinis an Fc containing protein, such as an Fc fused to a heterologousdomain (e.g., a non-Fc or non-antibody domain). A heterologous proteinmay be any protein, including an antigen binding portion of an antibodyand derivatives thereof, e.g., Fabs, scFvs, bispecific scFvs, singledomain antibodies (“sdAbs”) (e.g., V_(H)H or camelid antibodies andV_(NAR)s), diabodies (dAbs), single chain diabodies (scDb), Darpins,anticalins, and fibronectin based scaffolds, such as ¹⁰Fn3, Fibcons andTencons. A full length antibody may also be refolded as describedherein. Heterologous proteins linked to Fc may also be unrelated toantibodies and may be, e.g., TNFR.

Fibronectin Based Scaffolds

As used herein, a “fibronectin based scaffold” or “FBS” protein ormoiety refers to proteins or moieties that are based on a fibronectintype III (“Fn3”) repeat. Fn3 is a small (about 10 kDa) domain that hasthe structure of an immunoglobulin (Ig) fold (i.e., an Ig-likeβ-sandwich structure, consisting of seven β-strands and six loops).Fibronectin has 18 Fn3 repeats, and while the sequence homology betweenthe repeats is low, they all share a high similarity in tertiarystructure. Fn3 domains are also present in many proteins other thanfibronectin, such as adhesion molecules, cell surface molecules, e.g.,cytokine receptors, and carbohydrate binding domains. For reviews seeBork & Doolittle, Proc Natl Acad Sci USA 89(19):8990-4 (1992); Bork etal., J Mol Biol. 242(4):309-20 (1994); Campbell & Spitzfaden, Structure2(5):333-7 (1994); Harpez & Chothia, J Mol Biol. 238(4):528-39 (1994)).The term “fibronectin based scaffold” protein or moiety is intended toinclude scaffolds based on Fn3 domains from these other proteins (i.e.,non fibronectin molecules).

An example of fibronectin-based scaffold proteins are Adnectins(Adnexus, a wholly owned subsidiary of Bristol-Myers Squibb). It hasbeen shown that the CDR-like loop regions of the fibronectin basedscaffolds can be modified to evolve a protein capable of binding to anycompound of interest. For example, U.S. Pat. No. 7,115,396 describes Fn3domain proteins wherein alterations to the BC, DE, and FG loops resultin high affinity TNFα binders. U.S. Pat. No. 7,858,739 describes Fn3domain proteins wherein alterations to the BC, DE, and FG loops resultin high affinity VEGFR2 binders.

An Fn3 domain is small, monomeric, soluble, and stable. It lacksdisulfide bonds and, therefore, is stable under reducing conditions. Fn3domains comprise, in order from N-terminus to C-terminus, a beta orbeta-like strand, A; a loop, AB; a beta or beta-like strand, B; a loop,BC; a beta or beta-like strand, C; a loop, CD; a beta or beta-likestrand, D; a loop, DE; a beta or beta-like strand, E; a loop, EF; a betaor beta-like strand, F; a loop, FG; and a beta or beta-like strand, G.The seven antiparallel β-strands are arranged as two beta sheets thatform a stable core, while creating two “faces” composed of the loopsthat connect the beta or beta-like strands. Loops AB, CD, and EF arelocated at one face (“the south pole”) and loops BC, DE, and FG arelocated on the opposing face (“the north pole”). Any or all of loops AB,BC, CD, DE, EF and FG may participate in ligand binding.

In exemplary embodiments, the ligand binding fibronectin based scaffoldmoieties described herein are based on the tenth fibronectin type IIIdomain, i.e., the tenth module of Fn3 (¹⁰Fn3). The amino acid sequenceof wild-type human ¹⁰Fn3 (with N-terminal tail (in italics)) is setforth in SEQ ID NO: 1:

(SEQ ID NO: 1) VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPS Q(the AB, CD and EF loops are underlined; the BC, FG, and DE loops areemphasized in bold; the β-strands are located between each of the loopregions; and the N-terminal and C-terminal regions are shown initalics). Wild-type ¹⁰Fn3 without the tail set forth in italics in SEQID NO: 1 is provided as SEQ ID NO: 5.

In some embodiments, the AB loop corresponds to residues 14-17, the BCloop corresponds to residues 23-31, the CD loop corresponds to residues37-47, the DE loop corresponds to residues 51-56, the EF loopcorresponds to residues 63-67, and the FG loop corresponds to residues75-87 of SEQ ID NO: 1. The BC, DE and FG loops align along one face ofthe molecule, i.e. the “north pole”, and the AB, CD and EF loops alignalong the opposite face of the molecule, i.e. the “south pole”. In SEQID NO: 1, β-strand A corresponds to residues 8-13, β-strand Bcorresponds to residues 18-22, β-strand C corresponds to residues 32-36,beta strand D corresponds to residues 48-50, β-strand E corresponds toresidues 57-62, β-strand F corresponds to residues 68-74, and β-strand Gcorresponds to residues 88-92. The β-strands are connected to each otherthrough the corresponding loop, e.g., strands A and B are connected vialoop AB in the formation β-strand A, loop AB, β-strand B, etc. TheN-terminal and/or C-terminal regions of SEQ ID NO: 1 (italicized above),may be removed or altered to generate a molecule retaining biologicalactivity and comprising, e.g., an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2-16. In certain embodiments, the first8 amino acid residues of SEQ ID NO: 1 and/or the last 7 amino acidresidues of SEQ ID NO: 1 (i.e., amino acid residues 1-8 and 95-101 ofSEQ ID NO: 1, respectively) may be removed or altered to generate apolypeptide comprising the amino acid sequence of SEQ ID NO: 4.

As described above, amino acid residues corresponding to residues 14-17,23-31, 37-47, 51-56, 63-67 and 75-87 of SEQ ID NO: 1 define the AB, BC,CD, DE, EF and FG loops, respectively. However, it should be understoodthat not every residue within a loop region needs to be modified inorder to achieve a ¹⁰Fn3 binding domain having strong affinity for adesired target. Additionally, insertions and deletions in the loopregions may also be made while still producing high affinity ¹⁰Fn3binding domains.

Accordingly, in some embodiments, one or more loops selected from AB,BC, CD, DE, EF and FG may be extended or shortened in length relative tothe corresponding loop in wild-type human ¹⁰Fn3. In any givenpolypeptide, one or more loops may be extended in length, one or moreloops may be reduced in length, or combinations thereof. In someembodiments, the length of a given loop may be extended by 2-25, 2-20,2-15, 2-10, 2-5, 5-25, 5-20, 5-15, 5-10, 10-25, 10-20, or 10-15 aminoacids. In some embodiments, the length of a given loop may be reduced by1-15, 1-11, 1-10, 1-5, 1-3, 1-2, 2-10, or 2-5 amino acids. Inparticular, the FG loop of ¹⁰Fn3 is 13 residues long, whereas thecorresponding loop in antibody heavy chains ranges from 4-28 residues.To optimize antigen binding in polypeptides relying on the FG for targetbinding, therefore, the length of the FG loop of ¹⁰Fn3 may be altered inlength as well as in sequence to obtain the greatest possibleflexibility and affinity in target binding.

In some embodiments, one or more residues of the integrin-binding motif“arginine-glycine-aspartic acid” (RGD) (amino acids 78-80 of SEQ IDNO: 1) may be substituted so as to disrupt integrin binding. In someembodiments, the FG loop of the polypeptides provided herein does notcontain an RGD integrin binding site. In one embodiment, the RGDsequence is replaced by a polar amino acid-neutral amino acid-acidicamino acid sequence (in the N-terminal to C-terminal direction). Inanother embodiment, the RGD sequence is replaced with SGE. In yetanother embodiment, the RGD sequence is replaced with RGE (see, e.g.,SEQ ID NO: 16).

As used herein, a “¹⁰Fn3 domain” or “¹⁰Fn3 moiety” refers to wild-type¹⁰Fn3 (e.g., comprising one of SEQ ID NOs: 1-8, 10, 12, 14 or 16) andbiologically active variants thereof, e.g., biologically active variantsthat specifically bind to a target, such as a target protein andbiologically active variants having SEQ ID NO: 9, 11, 13 or 15. A wildtype ¹⁰Fn3 domain may comprise one of the amino acid sequenced set forthin SEQ ID NO: 1-8, 10, 12, 14 and 16. Biologically active variants of a¹⁰Fn3 domain include ¹⁰Fn3 domains that comprise at least, at most orabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 30, 35, 40 or 45 amino acid changes, i.e.,substitutions, additions or deletions, relative to a ¹⁰Fn3 domaincomprising any one of SEQ ID NOs: 1-16. A biologically active variant ofa ¹⁰Fn3 domain may also comprise, or comprise at most, 1-3, 1-5, 1-10,1-15, 1-10, or 1-25 amino acid changes relative to a ¹⁰Fn3domaincomprising any one of SEQ ID NOs: 1-16. In certain embodiments, abiologically active variant of a ¹⁰Fn3domain does not comprise more than1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1,2, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40 or 45 amino acid changes, i.e.,substitutions, additions or deletions, relative to an ¹⁰Fn3domaincomprising any one of SEQ ID NOs: 1-16. Amino acid changes may be in aloop region and/or in a strand. Exemplary degenerate ¹⁰Fn3 amino acidsequences are provided herein as SEQ ID NOs: 17-29.

In some embodiments, a fibronectin based scaffold moiety comprises a¹⁰Fn3 domain having at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, or 90% identity to a human ¹⁰Fn3 domain having an amino acidsequence selected from the group of sequence comprising SEQ ID NOs:1-16. In certain embodiments, the fibronectin based scaffold moietyprovided herein have at least 50% identity to an amino acid sequenceselected from the group of amino acid sequences comprising SEQ ID NO:1-16. In other embodiments, the fibronectin based scaffold moiety has atleast 65% identity to an amino acid sequence selected from the group ofamino acid sequences comprising SEQ ID NO: 1-16. In certain embodiments,one or more of the loops will not be modified relative to the sequenceof the corresponding loop of the wild-type sequence and/or one or moreof the β-strands will not be modified relative to the sequence of thecorresponding β-strand of the wild-type sequence. In certainembodiments, each of the beta or beta-like strands of a ¹⁰Fn3 domain ina fibronectin based scaffold moiety may comprise, consist essentiallyof, or consist of an amino acid sequence that is at least 80%, 85%, 90%,95% or 100% identical to the sequence of a corresponding beta orbeta-like strand of SEQ ID NO: 1. Preferably, variations in the β-strandregions will not disrupt the stability of the polypeptide inphysiological conditions. In exemplary embodiments, the ¹⁰Fn3 domainbinds to a desired target with a K_(d) of less than 500 nM, 100 nM, 50nM, 10 nM, 5 nM, 1 nM, 500 pM, 100 pM or less. In some embodiments, the¹⁰Fn3 domain of a fibronectin based protein scaffold binds to a desiredtarget with a K_(d) between 1 pM and 1 μM, between 100 pM and 500 nM,between 1 nM and 500 nM, or between 1 nM and 100 nM. In exemplaryembodiments, the fibronectin based scaffold moiety binds specifically toa target that is not bound by a wild-type ¹⁰Fn3 domain, particularly thewild-type human ¹⁰Fn3 domain having, e.g., SEQ ID NO: 1-8, 10, 12, 14 or16.

In some embodiments, fusion proteins comprise a fibronectin basedscaffold moiety comprising a ¹⁰Fn3 domain, wherein the ¹⁰Fn3 domaincomprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; a loop, EF;and a loop, FG; and has at least one loop selected from loop AB, BC, CD,DE, EF and FG with an altered amino acid sequence relative to thesequence of the corresponding loop of the human ¹⁰Fn3 domain of SEQ IDNO: 1-16. In some embodiments, the BC, DE and FG loops are altered. Incertain embodiments, the AB, CD and EF loops are altered. In certainembodiments, the FG loop is the only loop that is altered. In otherembodiments, the CD and FG loops are both altered, and optionally, noother loops are altered. In certain embodiments, the CD and EF loops areboth altered, and optionally, no other loops are altered. In someembodiments, one or more specific scaffold alterations are combined withone or more loop alterations. By “altered” is meant one or more aminoacid sequence alterations relative to a template sequence (i.e., thecorresponding wild-type human fibronectin domain) and includes aminoacid additions, deletions, and substitutions.

In some embodiments, the fibronectin based scaffold moiety comprises a¹⁰Fn3 domain wherein the non loop regions comprise an amino acidsequence that is at least 80, 85, 90, 95, 98, or 100% identical to thenon-loop regions of SEQ ID NO: 1, wherein at least one loop selectedfrom AB, BC, CD, DE, EF and FG is altered. For example, in certainembodiments, the AB loop may have up to 4 amino acid substitutions, upto 10 amino acid insertions, up to 3 amino acid deletions, or acombination thereof the BC loop may have up to 10 amino acidsubstitutions, up to 4 amino acid deletions, up to 10 amino acidinsertions, or a combination thereof the CD loop may have up to 6 aminoacid substitutions, up to 10 amino acid insertions, up to 4 amino aciddeletions, or a combination thereof the DE loop may have up to 6 aminoacid substitutions, up to 4 amino acid deletions, up to 13 amino acidinsertions, or a combination thereof the EF loop may have up to 5 aminoacid substitions, up to 10 amino acid insertions, up to 3 amino aciddeletions, or a combination thereof and/or the FG loop may have up to 12amino acid substitutions, up to 11 amino acid deletions, up to 25 aminoacid insertions, or a combination thereof.

In certain embodiments, a fibronectin based scaffold moiety comprises anamino acid sequence that is at least 40%, 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 97%, 98%, or 99% identical to an amino acid sequence selectedfrom the group of sequences consisting of SEQ ID NOs: 1-16, and thefusion protein binds specifically to a target, e.g., with a K_(d) ofless than 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 100 pM orless. The proteins may comprise amino acid changes (or alterations) inone or more loops and one or more strands.

In certain embodiments, the fibronectin based scaffold moiety comprisesa ¹⁰Fn3 domain that is defined generally by following the sequence:

(SEQ ID NO: 17) VSDVPRD LEVVAA (X)_(u) LLISW (X)_(v) YRITY (X)_(w) FTV(X)_(x) ATISGL (X)_(y) YTITVYA (X)_(z) ISINY RT,

or by the sequence having SEQ ID NO: 18-29. In SEQ ID NOs: 17-29, the ABloop is represented by (X)_(u), the BC loop is represented by (X)_(v),the CD loop is represented by (X)_(w), the DE loop is represented by(X)_(x), the EF loop is represented by (X)_(y) and the FG loop isrepresented by X_(z). X represents any amino acid and the subscriptfollowing the X represents an integer of the number of amino acids. Inparticular, u, v, w, x, y and z may each independently be anywhere from2-20, 2-15, 2-10, 2-8, 5-20, 5-15, 5-10, 5-8, 6-20, 6-15, 6-10, 6-8,2-7, 5-7, or 6-7 amino acids. The sequences of the beta strands(underlined) may have anywhere from 0 to 10, from 0 to 8, from 0 to 6,from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1substitutions, deletions or additions across all 7 scaffold regionsrelative to the corresponding amino acids shown in SEQ ID NOs: 17-29. Insome embodiments, the sequences of the beta strands may have anywherefrom 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0to 3, from 0 to 2, or from 0 to 1 substitutions, e.g., conservativesubstitutions, across all 7 scaffold regions relative to thecorresponding amino acids shown in SEQ ID NO: 17-29. In certainembodiments, the hydrophobic core amino acid residues (bolded residuesin SEQ ID NO: 17 above) are fixed, and any substitutions, conservativesubstitutions, deletions or additions occur at residues other than thehydrophobic core amino acid residues. In some embodiments, thehydrophobic core residues of the polypeptides provided herein have notbeen modified relative to the wild-type human ¹⁰Fn3 domain (e.g., SEQ IDNO: 1 or 5).

In some embodiments, the amino acid sequences of the N-terminal and/orC-terminal regions of a fibronectin based scaffold moiety may bemodified by deletion, substitution or insertion relative to the aminoacid sequences of the corresponding regions of ¹⁰Fn3 domains comprisingone of SEQ ID NOs: 1-16). In some embodiments, the first eight (i.e.,residues 1-8) and the last seven amino acids (i.e., residues 95-101) ofSEQ ID NO: 1 are deleted, generating a ¹⁰Fn3 domain having the aminoacid sequence of SEQ ID NO: 4. Additional sequences may also be added tothe N- or C-terminus of a ¹⁰Fn3 domain having the amino acid sequence ofany one of SEQ ID NOs: 1-16. For example, in some embodiments, theN-terminal extension consists of an amino acid sequence selected fromthe group consisting of: M, MG, and G.

In certain embodiments, the amino acid sequence of the first 1, 2, 3, 4,5, 6, 7, 8 or 9 residues of SEQ ID NO: 1 may be modified or deleted inthe polypeptides provided herein relative to the sequence of thecorresponding amino acids in the wild-type human ¹⁰Fn3 domain having SEQID NO: 1. In exemplary embodiments, the amino acids corresponding toamino acids 1-8 of SEQ ID NO: 1 are replaced with an alternativeN-terminal region having from 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2,or 1 amino acids in length. Exemplary alternative N-terminal regionsinclude (represented by the single letter amino acid code) M, MG, G,MGVSDVPRDL (SEQ ID NO: 30) and GVSDVPRDL (SEQ ID NO: 31), or N-terminaltruncations of any one of SEQ ID NOs: 30 and 31. Other suitablealternative N-terminal regions include, for example, X_(n)SDVPRDL (SEQID NO: 32), X_(n)DVPRDL (SEQ ID NO: 33), X_(n)VPRDL (SEQ ID NO: 34),X_(n)PRDL (SEQ ID NO: 35), X_(n)RDL (SEQ ID NO: 36), X_(n)DL (SEQ ID NO:37), or X_(n)L, wherein n=0, 1 or 2 amino acids, wherein when n=1, X isMet or Gly, and when n=2, X is Met-Gly. When a Met-Gly sequence is addedto the N-terminus of a ¹⁰Fn3 domain, the M will usually be cleaved off,leaving a G at the N-terminus. In other embodiments, the alternativeN-terminal region comprises the amino acid sequence MASTSG (SEQ ID NO:38).

In certain embodiments, the amino acid sequence corresponding to aminoacids 93-101, 94-101, 95-101, 96-101, 97-101, 98-101, 99-101, 100-101,or 101 of SEQ ID NO: 1 are deleted or modified in the polypeptidesprovided herein relative to the sequence of the corresponding aminoacids in the wild-type human ¹⁰Fn3 domain (SEQ ID NO: 1). In exemplaryembodiments, the amino acids corresponding to amino acids 95-101 of SEQID NO: 1 are replaced with an alternative C-terminal region having from1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids in length.Specific examples of alternative C-terminal region sequences include,for example, polypeptides comprising, consisting essentially of, orconsisting of, EIEK (SEQ ID NO: 39), EGSGC (SEQ ID NO: 40), EIEKPCQ (SEQID NO: 41), EIEKPSQ (SEQ ID NO: 42), EIEKP (SEQ ID NO: 43), EIEKPS (SEQID NO: 44), EIEKPC (SEQ ID NO: 45), or HHHHHH (SEQ ID NO: 46). In someembodiments, the alternative C-terminal region comprises EIDK (SEQ IDNO: 47), and in particular embodiments, the alternative C-terminalregion is either EIDKPCQ (SEQ ID NO: 48) or EIDKPSQ (SEQ ID NO: 49).

In certain embodiments, a fibronectin based scaffold moiety comprises a¹⁰Fn3 domain having both an alternative N-terminal region sequence andan alternative C-terminal region sequence.

In certain embodiments, a fibronectin based scaffold moiety is based onan Fn3 repeat other than the 10^(th) repeat of the type III domain offibronectin, e.g., human fibronectin. For example, a fibronectin basedscaffold moiety may be similar to any of the other fibronectin type IIIrepeats, e.g., the 1^(st), 2^(nd), 3^(rd), 4^(th), 5^(th), 6^(th),7^(th), 8^(th), 9^(th), 11^(th), 12^(th), 13^(th), 14^(th), 15^(th),16^(th), 17^(th), and 18^(th) Fn3 repeats. In yet other embodiments, afibronectin based scaffold moiety may be from a molecule other thanfibronectin. Exemplary fibronectin based scaffold moieties may bederived from tenascin, a protein that is composed of 15 Fn3 domains withsimilar sequence similarities to one another as found in fibronectin.These repeats are described, e.g., in Jacobs et al. (2012) ProteinEngineering, Design & Selection 25:107. Based on the homology of therepeats in the fibronectin molecule and those in the tenascin molecule,artificial molecules based on these homologies have been created. Theconsensus amino acid sequences based on the homology of the domains inthe fibronectin molecule are referred to as Fibcon and FibconB(WO2010/093627 and Jacobs et al. (2012) supra) and those based on thehomology of the domains in the tenascin molecule are referred to asTencon. An exemplary Fibcon amino acid sequence comprises the followingamino acid sequence:MPAPTDLRFTNETPSSLLISWTPPRVQITGYIIRYGPVGSDGRVKEFTVPPSVSSATITGLKPGTEYTISVIALKDNQESEPLRGRVTTGG (FibconB; SEQ ID NO: 50), wherein loopAB consists of amino acids 13-16 (TPSS; SEQ ID NO: 51), loop BC consistsof amino acids 22-28 (TPPRVQI; SEQ ID NO: 52), loop CD consists of aminoacids 38-43 (VGSDGR; SEQ ID NO: 53), loop DE consists of amino acids51-54 (PSVS; SEQ ID NO: 54), loop EF consists of amino acids 60-64(GLKPG; SEQ ID NO: 55) and loop FG consist of amino acids 75-81(KDNQESEP; SEQ ID NO: 56). Another Fibcon amino acid sequence comprisesthe following amino acid sequence:LDAPTDLQVTNVTDTSITVSWTPPSATITGYRITYTPSNGPGEPKELTVPPSSTSVTITGITPGVEYVVSVYALKDNQESPPLVGTCTT (SEQ ID NO: 57; Jacobs et al., supra).

Tenascin derived Fn3 proteins include Tencons (WO2010/051274,WO2010/051310 and WO2011/137319, which are specifically incorporated byreference herein). An exemplary Tencon protein has the following aminoacid sequence: LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 58; Jacobs et al., supra,and WO2011/137319), wherein loop AB consists of amino acids 13-16 (TEDS;SEQ ID NO: 59, loop BC consists of amino acids 22-28 (TAPDAAF; SEQ IDNO: 60), loop CD consists of amino acids 38-43 (SEKVGE; SEQ ID NO: 61),loop DE consists of amino acids 51-54 (GSER; SEQ ID NO: 62), loop EFconsists of amino acids 60-64 (GLKPG; SEQ ID NO: 63) and loop FGconsists of amino acids 75-81 (KGGHRSN; SEQ ID NO: 64).

A Fibcon, FibconB or Tencon moiety, or target binding variants thereof,whether by themselves or linked to a heterologous moiety, e.g., an Fc,may be refolded as described herein. Fn3 domains from other proteins,e.g., cell surface hormone and cytokine receptors, chaperonins, andcarbohydrate-binding domains, may also be refolded as described herein,either on their own or as part of a fusion protein to, e.g., Fc.

Fibronectin based scaffold proteins or moieties are described, e.g., inWO2010/093627, WO2011/130324, WO2009/083804, WO2009/133208, WO02/04523,WO2012/016245, WO2009/023184, WO2010/051310, WO2011/020033,WO2011/051333, WO2011/051466, WO2011/092233, WO2011/100700,WO2011/130324, WO2011/130328, WO2011/137319, WO2010/051274,WO2009/086116, WO09/058379 and WO2013/067029 (all of which arespecifically incorporated by reference herein, in particular, thevarious types of molecules are specifically incorporated by referenceherein): any of the fibronectin based scaffold proteins or moietiesdescribed in these publications may be refolded as described herein.

In certain embodiments, a protein that may be refolded as describedherein is a multivalent protein that comprises two or more fibronectinbased scaffold moieties, e.g., ¹⁰Fn3 domains. For example, a multivalentfusion protein may comprise 2, 3 or more fibronectin based scaffoldmoieties, e.g., ¹⁰Fn3 domains, that are covalently associated. Inexemplary embodiments, the fusion protein is a bispecific or dimericprotein comprising two ¹⁰Fn3 domains.

Fc Domains

Proteins that may be refolded as described herein include fusionproteins that comprise an Fc portion fused to a heterologous portion. Insome aspects, the heterologous portion is a fibronectin based scaffold,e.g., an ¹⁰Fn3 domain, however, the heterologous portion may be anyother protein.

As used herein, “Fc portion” encompasses domains derived from theconstant region of an immunoglobulin, preferably a human immunoglobulin,including a fragment, analog, variant, mutant or derivative of theconstant region. Suitable immunoglobulins include IgG1, IgG2, IgG3,IgG4, and other classes such as IgA, IgD, IgE and IgM. The constantregion of an immunoglobulin is defined as a naturally-occurring orsynthetically-produced polypeptide homologous to the immunoglobulinC-terminal region, and can include a CH1 domain, a hinge, a CH2 domain,a CH3 domain, or a CH4 domain, separately or in combination. The term“Fc moiety” or “Fc domain” as used herein refers to any of thecombination of CH1, hinge, CH2, CH3 and CH4 domains. Thus, an “Fcdomain” or moiety may or may not comprise a hinge.

Shown below is the sequence of a human IgG1 immunoglobulin constantregion, and the relative position of each domain within the constantregion are indicated based on the EU numbering format:

(SEQ ID NO: 65) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.The core hinge sequence is underlined, and the CH1 region is italicized;the CH2 and CH3 regions are in regular text. It should be understoodthat the C-terminal lysine is optional. In certain embodiments, theC-terminal lysine of an IgG sequence may be removed or replaced with anon-lysine amino acid, such as alanine, to further increase the serumhalf-life of the Fc fusion protein.

In certain embodiments, an Fc fusion protein comprises a human hinge,CH2 and CH3 domains, and may have the following amino acid sequence:

(SEQ ID NO: 66) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,wherein the core hinge sequence is underlined and the CH2 and CH3regions are in regular text.

In certain embodiments, an Fc fusion protein comprises a CH2 and a CH3region of a human IgG1 as shown below:

VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 67). Itshould be understood that the glycine and lysine at the end of a CH3domain are optional.

Fc fusion proteins may also comprise an Fc domain that is at least 50%,60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ IDNOs: 65-67. An Fc fusion protein may also comprise an Fc domain havingat least 50, 100, or 150 contiguous amino acids of SEQ ID NOs: 65-67. Fcfusion proteins may also comprise an Fc domain having from 50-100,50-150, or 100-150 contiguous amino acids of any one of SEQ ID NOs:65-67. Fc fusion proteins may comprise an Fc domain comprising any oneof SEQ ID NOs: 65-67 with from 1-5, 1-10, 1-15, 1-20, or 1-25substitutions, e.g., conservative substitutions.

The Fc domain may be a naturally occurring Fc sequence, includingnatural allelic or splice variants. Alternatively, an Fc domain may be anon-naturally occurring Fc domain, e.g., a hybrid domain comprising aportion of an Fc domain from two or more different Ig isotypes, forexample, an IgG2/IgG4 hybrid Fc domain. In exemplary embodiments, the Fcdomain is derived from a human immunoglobulin molecule. Alternatively,the Fc domain may be a humanized or deimmunized version of an Fc domainfrom a non-human animal, including but not limited to mouse, rat,rabbit, camel, llama, dromedary and monkey.

In certain embodiments, the Fc domain is a variant Fc sequence, e.g., anFc sequence that has been modified (e.g., by amino acid substitution,deletion and/or insertion) relative to a parent Fc sequence (e.g., anunmodified Fc polypeptide that is subsequently modified to generate avariant), to provide desirable structural features and/or biologicalactivity.

For example, one may make modifications in the Fc region in order togenerate an Fc variant that (a) has increased or decreasedantibody-dependent cell-mediated cytotoxicity (ADCC), (b) increased ordecreased complement mediated cytotoxicity (CDC), (c) has increased ordecreased affinity for C1q and/or (d) has increased or decreasedaffinity for a Fc receptor relative to the parent Fc. Such Fc regionvariants will generally comprise at least one amino acid modification inthe Fc region. Combining amino acid modifications is thought to beparticularly desirable. For example, the variant Fc region may includetwo, three, four, five, etc substitutions therein, e.g., of the specificFc region positions identified herein. Proteins comprising Fcs that aremutated to modify the biological activity of the Fc may be refolded asdescribed herein. Exemplary Fc mutants are described, e.g., inWO97/34631; WO96/32478; U.S. Pat. No. 5,624,821; U.S. Pat. No.5,648,260; U.S. Pat. No. 6,194,551; WO 94/29351; WO00/42072; U.S. Pat.No. 5,624,821; U.S. Pat. No. 6,277,375; U.S. Pat. No. 6,737,056; U.S.Pat. No. 6,194,551; U.S. Pat. No. 7,317,091; U.S. Pat. No. 8,101,720;PCT Patent Publications WO 00/42072; WO 01/58957; WO 02/06919; WO04/016750; WO 04/029207; WO 04/035752; WO 04/074455; WO 04/099249; WO04/063351; WO 05/070963; WO 05/040217; WO 05/092925; WO 06/020114; andStrohl, 2009, Current Opinion in Biotechnology 20:685-691; U.S. Pat. No.6,277,375; Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216; Hintonet al. 2006 Journal of Immunology 176:346-356; Dall Acqua et al. Journalof Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006, Journal ofBiological Chemistry 281:23514-23524; Yeung et al., 2010, J Immunol,182:7663-7671; WO88/07054; WO88/07089; U.S. Pat. No. 6,277,375;WO99/051642; WO01/058957; WO2003/074679; WO2004/029207; U.S. Pat. No.7,317,091 and WO2004/099249.

Exemplary variant Fcs are set forth as SEQ ID NOs: 68-86. In someaspects, an Fc fusion protein described herein comprises an Fc domainhaving at least 50, 100, or 150 contiguous amino acids of any one of SEQID NOs: 68-86. In other embodiments, an Fc fusion protein describedherein comprises an Fc domain having from 50-100, 50-150, or 100-150contiguous amino acids of SEQ ID NOs: 68-86. In yet other embodiments,an Fc fusion protein described herein comprises an Fc domain comprisingSEQ ID NOs: 68-86 with from 1-5, 1-10, 1-15, 1-20, or 1-25substitutions, e.g., conservative substitutions.

Fc fusion proteins may contain an immunoglobulin hinge region. The hingeregion may be derived from antibodies belonging to any of theimmunoglobulin classes, i.e. IgA, IgD, IgE, IgG, or IgM. In certainembodiments, the hinge region is derived from any of the IgG antibodysubclasses, i.e. IgG1, IgG2, IgG3, and IgG4. In some embodiments, thehinge region may further include residues derived from the CH1 and CH2regions that flank the core hinge sequence, as discussed further below.

In certain embodiments, a hinge contains a free cysteine residue that iscapable of forming a disulfide bond with another monomer to form adimer. The hinge sequence may naturally contain a cysteine residue, ormay be engineered to contain one or more cysteine residues.

In certain embodiments, the Fc fusion proteins comprise a hinge regionderived from a human IgG1. In some embodiments, the hinge regioncomprises the core hinge residues DKTHTCPPCPAPELLG (SEQ ID NO: 87) ofIgG1, which corresponds to positions 221-236 according to EU numbering.

In certain embodiments, the hinge sequence may include substitutionsthat confer desirable pharmacokinetic, biophysical, and/or biologicalproperties. Some exemplary hinge sequences include

(SEQ ID NO: 88; core hinge region underlined) EPKSSDKTHTCPPCPAPELLGGPS,(SEQ ID NO: 89; core hinge region underlined) EPKSSDKTHTCPPCPAPELLGGSS,(SEQ ID NO: 90; core hinge region underlined) EPKSSGSTHTCPPCPAPELLGGSS,(SEQ ID NO: 91; core hinge region underlined) DKTHTCPPCPAPELLGGPS, and(SEQ ID NO: 92, core hinge region underlined) DKTHTCPPCPAPELLGGSS.In one embodiment, the hinge sequence is a derivative of an IgG1 hingecomprising a P122S substitution (EU numbering 238) (e.g., the Prolineresidue at position 122 in SEQ ID NO: 22 is substituted with serine).The P122S substitution ablates Fc effector function and is exemplifiedby the hinges having any one of SEQ ID NOs: 25, 26, and 28. In anotherembodiment, the hinge sequence is a derivative of an IgG1 hingecomprising D104G and K105S substitutions (EU numbering 221-222). TheD104G and K105S substitutions remove a potential cleavage site andtherefore increase the protease resistance of the fusion molecule. Ahinge having D104G and K105S substitutions is exemplified in SEQ ID NO:26. In another embodiment, the hinge sequence is a derivative of an IgG1hinge comprising a C103S substitution (EU numbering 220). The C103Ssubstitution prevents improper cysteine bond formation in the absence ofa light chain. Hinges having a C103S substitution are exemplified by SEQID NOs: 24-26.

Fc fusion proteins may comprise a hinge sequence that comprises,consists essentially of, or consists of an amino acid sequence that isat least 50%, 60%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% tothat of any hinge described herein, e.g., a hinge having SEQ ID NOs:88-92, or comprises, consists essentially of, or consists of an aminoacid sequence of any hinge described herein, e.g., one of SEQ ID NOs:88-92. Fc fusion proteins may comprise a hinge portion comprises atleast or at most 2, 5, 10, 12, 15, 18 or 20 contiguous amino acidresidues from any of SEQ ID NOs: 88-92, or a sequence comprising from1-5, 1-10, 1-15, 1-20, 2-5, 2-10, 2-15, 2-20, 5-10, 5-15, 5-20, 10-15,10-20, or 15-20 contiguous amino acid residues from any of SEQ ID NOs:88-92. In exemplary embodiments, the hinge sequence comprises a cysteineresidue.

In certain embodiments, an Fc fusion protein does not comprise a hinge.For example, an Fc fusion protein may comprise an Fc domain linked to aheterologous protein, e.g., in the Fc-X or X-Fc format, withoutcomprising a hinge or a core hinge. In one example, an Fc fusion proteindoes not comprise the sequence EPKSSDKTHTCPPCP (SEQ ID NO: 93) or avariant thereof.

In certain embodiments, an Fc fusion protein does not comprise a linker.For example, an Fc fusion protein may comprise an Fc domain that islinked directly to a heterologous protein, e.g., a ¹⁰Fn3 protein withoutan intervening sequence. In certain embodiments, there may be 1, 2, 3, 4or 5 amino acids (e.g., from 1-5 or 1-10 amino acids) between the Fcdomain and the heterologous protein. Such Fc fusion proteins may be X-Fc(the heterologous protein is linked at the N-terminus of the Fc) or Fc-X(the heterologous protein is linked at the C-terminus of the Fc) fusionproteins, wherein X is the heterologous protein, and wherein X and Fcare directly linked to each other.

In certain embodiments, an Fc fusion protein comprises neither a hingenor a linker.

In certain embodiments, an Fc fusion protein is a dimer, wherein eachmonomer comprises a fusion protein (a homodimer). In certainembodiments, an Fc fusion protein is a heterodimer comprising, e.g., amonomer that comprises an Fc fusion protein and a monomer that comprisesan Fc that is not linked to another moiety. The Fc portion of a monomermay comprise one or more amino acid modifications (or mutations)relative to a wild type Fc that favor dimer, e.g., heterodimer,formation with another Fc. For example, an Fc of a dimer may comprise a“hole” and the other Fc of the dimer may comprise a “bump” or “knob,” asdescribed, e.g., in WO96/027011; U.S. Pat. No. 5,731,168 and U.S. Pat.No. 5,821,333. Other modifications, such as electrostatic modificationsmay be used to enhance dimer formation. Exemplary modifications aredescribed, e.g., in WO2007/110205; WO2009/089004 and WO2010/129304. Suchchanges are particularly useful for enhancing the association of twoheterologous monomers to form a dimer, such as a dimer that comprises amonomer comprising an Fc fusion protein and a monomer comprising an Fcthat is different from the Fc fusion protein, e.g., by the lack of aheterologous protein.

In certain embodiments, an Fc fusion protein comprises a monomercomprising the structure X-Fc and a monomer comprising the structureFc-X (or Fc-Y). An Fc fusion protein may also comprise two monomers,each comprising the structure X-Fc-X (a “quad” structure), as used,e.g., in Examples 1-3. An Fc fusion protein may also comprise twomonomers, each comprising the structure X-Fc-Y, or one monomercomprising the structure X-Fc-Y and a monomer comprising the structureY-Fc-X. Each monomer may optionally comprise a linker and optionallycomprise a hinge.

An Fc fusion protein may comprise a single chain Fc (scFc), wherein thefirst and the second Fc domain (or the first and the second hinge-Fcdomains) are linked through a linker. In one embodiment, a scFccomprises in N- to C-terminal order a first CH2 domain, which first CH2domain is linked to a first CH3 domain, which CH3 domain is linked to anFc linker, which Fc linker is linked the a second CH2 domain, whichsecond CH2 domain is linked to a second CH3 domain, wherein the firstand the second CH2 and CH3 domains associate to form a dimeric Fc. AnscFc may comprise in N- to C-terminal order a first hinge, which firsthinge is linked to a first CH2 domain, which first CH2 domain is linkedto a first CH3 domain, which first CH3 domain is linked to an Fc linker,which Fc linker is linked to a second hinge, which second hinge islinked to a second CH2 domain, which second CH2 domain is linked to asecond CH3 domain, wherein the first and the second hinges, CH2 domainsand CH3 domains associate to form a dimeric Fc. scFcs are described,e.g., in WO2008/131242, WO2008/143954 and WO2008/012543.

Exemplary Linkers for Connecting a Heterologous Protein to an Fc Moiety

Any linker may be used for covalently linking a heterologous protein,e.g., a fibronectin based scaffold moiety, to an Fc moiety, providedthat the linker allows the fusion protein comprising the heterologousprotein to properly fold and be biologically active. For example, thefusion protein should be able to bind efficiently to its target and mayhave a long half-life in serum relative to the heterologous protein thatis not linked to an Fc. A linker is also preferably essentially notimmunogenic and not reactive with other proteins (i.e., chemicallyinert).

A linker may be from 1-6, 1-10, 1-15, 1-20, 1-25, 1-30, 1-35, 1-40,1-45, 1-50, 5-10, 5-15, 5-15, 5-20, 5-25, 5-30, 5-35, 5-40, 5-45, or5-50 amino acids long.

Exemplary linkers may comprise, consist of, or consist essentially of GSlinkers, e.g., (GS)₁, (GS)₂, (GS)₃, (GS)₄, (GS)₅, (GS)₆, (GS)₇, (GS)₈,(GS)₉ or (GS)₁₀. Linkers may also comprise, consist of, or consistessentially of G₄S linkers, e.g., (G₄S)₁, (G₄S)₂, (G₄S)₃, (G₄S)₄ or(G₄S)₅. Additional exemplary linkers are provided in WO2012/142515.

Exemplary Fc Fusion Proteins that May be Refolded

Fusion proteins comprising a heterologous moiety X, e.g., ¹⁰Fn3, and anFc moiety are collectively referred to herein as X/Fc fusions regardlessof whether they contain a linker or a hinge and regardless oforientation (the “/” indicates that it covers both orientations, i.e.,where the Fc is N-terminal or where the Fc is C-terminal to X).

In certain embodiments, an Fc is linked directly to X, i.e., without oneor more intervening amino acid (e.g., without a linker). In certainembodiment, an Fc is linked indirectly to X, i.e., with one or moreintervening amino acids, e.g., a linker.

Exemplary fusion proteins are as follows, and as shown in the N- toC-terminal order:

X-hinge-CH2-CH3; X-linker-hinge-CH2-CH3; X—CH2-CH3; X-linker-CH2-CH3;hinge-CH2-CH3-X; hinge-CH2-CH3-linker-X; CH2-CH3-Fc; CH2-CH3-linker-X,wherein X is a heterologous protein relative to the Fc portion. Ineither orientation, the X/Fc fusion proteins described herein mayfurther contain an N-terminal methionine and/or a leader sequence (e.g.,for expression in mammalian cells).

In certain embodiments, a fusion protein comprises (i) a fibronectinbased scaffold moiety comprising an amino acid sequence that is at leastabout 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to anyone of SEQ ID NOs: 1-29; and (ii) an Fc moiety, e.g., comprising anamino acid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%,97%, 98%, or 99% identical to SEQ ID NO: 65-86, wherein the fusionprotein binds specifically to a target (e.g., with a K_(d) of less than500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 100 pM or less) thatis not bound by a wild-type fibronectin based scaffold moiety (e.g., SEQID NOs: 1-8, 10, 12, 14 or 16). In certain embodiments, a fusion proteincomprises (i) a fibronectin based scaffold moiety comprising an aminoacid sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%,98%, or 99% identical to any one of SEQ ID NOs: 1-29; (ii) an Fc moiety,e.g., comprising an amino acid sequence that is at least about 70%, 75%,80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 65-86; and(iii) a linker that covalently links the fibronectin based scaffoldmoiety to the Fc moiety, wherein the fusion protein binds specificallyto a target (e.g., with a K_(d) of less than 500 nM, 100 nM, 50 nM, 10nM, 5 nM, 1 nM, 500 pM, 100 pM or less) that is not bound by a wild-typefibronectin based scaffold moiety (e.g., SEQ ID NOs: 1-8, 10, 12, 14 or16). In certain embodiments, a fusion protein comprises (i) afibronectin based scaffold moiety comprising an amino acid sequence thatis at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 1-29, (ii) an Fc moiety, e.g.,comprising an amino acid sequence that is at least about 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 65-86; and (iii)a linker that covalently links the fibronectin based scaffold moiety tothe Fc moiety, wherein the linker comprises 1-10 amino acids, such as 6amino acids, and wherein the fusion protein binds specifically to atarget (e.g., with a K_(d) of less than 500 nM, 100 nM, 50 nM, 10 nM, 5nM, 1 nM, 500 pM, 100 pM or less) that is not bound by a wild-typefibronectin based scaffold moiety (e.g., SEQ ID NOs: 1-8, 10, 12, 14 or16). Exemplary fibronectin based scaffold moieties linked to an Fc aredisclosed in WO2012/142515.

Also provided herein are protein compositions, e.g., compositionscomprising one or more protein in one of the solutions or buffersdescribed herein. For example, provided herein are compositionscomprising a protein comprising at least two cysteines (wherein, e.g.,the protein is a dimer comprising one cysteine on each dimer) that forma disulfide bond under appropriate conditions, and water, wherein thecomposition does not comprise a significant amount of buffer or adenaturing agent and optionally does not comprise a reducing agent. Alsoprovided herein are compositions comprising a suspension of denaturedproteins, wherein at least some proteins comprise at least two cysteinesthat form a disulfide bond under appropriate conditions, and wherein thecomposition does not comprise a buffer or a denaturing agent andoptionally does not comprise a reducing agent. Further provided hereinare compositions comprising a protein comprising at least two cysteinesthat form a disulfide bond under appropriate conditions, and asolubilization buffer having a pH in the range of pH 10 to 13, whereinthe composition does not comprise a denaturing agent and optionally doesnot comprise a reducing agent. Also provided are compositions comprisinga protein comprising at least two cysteines that form a disulfide bondunder appropriate conditions, and a refold buffer having a pH in therange of pH 9 to 11 and an oxidizing agent, wherein the composition doesnot comprise a reducing agent other than a reducing agent that part ofan oxidizing agent that is present in the composition. The proteinconcentration in any of these compositions may be at least 20 mg/ml, 15mg/ml, 10 mg/ml, 5 mg/ml or 1 mg/ml. The compositions may comprise atleast 70%, 80%, 90%, 95%, 97%, 98% or 99% of the protein of interest,e.g., a fibronectin based scaffold moiety linked to an Fc, relative tothe total amount (e.g., in mg/ml) of protein in the composition. In thecomposition comprising refold buffer, refolded protein may constitute atleast 70%, 80%, 90%, 95%, 97%, 98% or 99% of the protein in the sample.

Protein Synthesis

Proteins that can be refolded as described herein may be synthesizedaccording to any method known in the art. Suitable host cells includeprokaryotes, yeast, mammalian cells, or bacterial cells. Suitablebacteria include gram negative or gram positive organisms, for example,E. coli or Bacillus spp. Yeast, preferably from the Saccharomycesspecies, such as S. cerevisiae, may also be used for production ofpolypeptides. Various mammalian or insect cell culture systems can alsobe employed to express recombinant proteins. Baculovirus systems forproduction of heterologous proteins in insect cells are reviewed byLuckow and Summers, (Bio/Technology, 6:47, 1988). Purified proteins maybe prepared by culturing suitable host/vector systems to express therecombinant proteins. Expressed proteins, e.g., fibronectin basedscaffold proteins, may then be purified from culture media or cellextracts.

Proteins may be synthesized chemically, enzymatically or recombinantly.Proteins may also be produced using cell-free translation systems. Forsuch purposes the nucleic acids encoding the fusion protein must bemodified to allow in vitro transcription to produce mRNA and to allowcell-free translation of the mRNA in the particular cell-free systembeing utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system). For chemical synthesis, see, e.g., the methodsdescribed in Solid Phase Peptide Synthesis, 2nd ed., 1984, The PierceChemical Co., Rockford, Ill.).

Codon usage may be selected so as to improve expression in a cell. Suchcodon usage will depend on the cell type selected. Specialized codonusage patterns have been developed for E. coli and other bacteria, aswell as mammalian cells, plant cells, yeast cells and insect cells. Seefor example: Mayfield et al., Proc Natl Acad Sci USA. 2003 Jan. 21;100(2):438-42; Sinclair et al. Protein Expr Purif. 2002 October;26(1):96-105; Connell N D. Curr Opin Biotechnol. 2001 October;12(5):446-9; Makrides et al. Microbiol Rev. 1996 September;60(3):512-38; and Sharp et al. Yeast. 1991 October; 7(7):657-78.

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F.Ausubel et al., Current Protocols in Molecular Biology (Green Publishingand Wiley-Interscience: New York, 1987) and periodic updates, hereinincorporated by reference. The DNA encoding the polypeptide is operablylinked to suitable transcriptional or translational regulatory elementsderived from prokaryotic, mammalian, viral, or insect genes. Suchregulatory elements include a transcriptional promoter, an optionaloperator sequence to control transcription, a sequence encoding suitablemRNA ribosomal binding sites, and sequences that control the terminationof transcription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants are additionally incorporated.

The proteins may comprise a signal sequence or other polypeptide havinga specific cleavage site at the N-terminus of the mature protein orpolypeptide. The heterologous signal sequence selected preferably is onethat is recognized and processed (i.e., cleaved by a signal peptidase)by the host cell. For prokaryotic host cells that do not recognize andprocess a native signal sequence, the signal sequence may be substitutedby a prokaryotic signal sequence selected, for example, from the groupof the alkaline phosphatase, penicillinase, lpp, or heat-stableenterotoxin II leaders. For yeast secretion the native signal sequencemay be substituted by, e.g., the yeast invertase leader, a factor leader(including Saccharomyces and Kluyveromyces alpha-factor leaders), oracid phosphatase leader, the C. albicans glucoamylase leader, or thesignal described in PCT Publication No. WO90/13646. In mammalian cellexpression, mammalian signal sequences as well as viral secretoryleaders, for example, the herpes simplex gD signal, are available. TheDNA for such precursor regions may be ligated in reading frame to DNAencoding the protein.

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid encoding the fusion protein. Promoters suitable for use withprokaryotic hosts include the phoA promoter, beta-lactamase and lactosepromoter systems, alkaline phosphatase, a tryptophan (trp) promotersystem, and hybrid promoters such as the tac promoter. However, otherknown bacterial promoters are suitable. Promoters for use in bacterialsystems also will contain a Shine-Dalgarno (S.D.) sequence operablylinked to the DNA encoding the protein.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the fusion proteins. Examples ofprotein tags include but are not limited to a histidine tag, a FLAG tag,a myc tag, an HA tag, or a GST tag. Appropriate cloning and expressionvectors for use with bacterial, fungal, yeast, and mammalian cellularhosts can be found in Cloning Vectors: A Laboratory Manual, (Elsevier,New York, 1985), the relevant disclosure of which is hereby incorporatedby reference.

The expression construct may be introduced into the host cell using amethod appropriate to the host cell, as will be apparent to one of skillin the art. A variety of methods for introducing nucleic acids into hostcells are known in the art, including, but not limited to,electroporation; transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (where thevector is an infectious agent).

Expression in bacterial cells may be conducted essentially as follows orwith certain variations therein. DNA encoding a protein of interest,e.g., a ¹⁰Fn3/Fc protein, is inserted into the pET9d (EMD Bioscience,San Diego, Calif.) vector and are expressed in E. coli HMS 174 cells.Twenty ml of an inoculum culture (generated from a single plated colony)is used to inoculate 1 liter of LB medium containing 50 μg/mlcarbenicillin and 34 μg/ml chloromphenicol. The culture is grown at 37°C. until A₆₀₀ 0.6-1.0. After induction with 1 mMisopropyl-β-thiogalactoside (IPTG) the culture is grown for 4 hours at30° C. and is harvested by centrifugation for 30 minutes at 10,000 g at4° C. Cell pellets are frozen at −80° C. The cell pellet is resuspendedin 25 ml of lysis buffer (20 mM NaH₂PO₄, 0.5 M NaCl, 1× CompleteProtease Inhibitor Cocktail-EDTA free (Roche), 1 mM PMSF, pH 7.4) usingan Ultra-turrax homogenizer (IKA works) on ice. Cell lysis is achievedby high pressure homogenization (18,000 psi) using a Model M-110SMICROFLUIDIZER® (Microfluidics). The insoluble fraction is separated bycentrifugation for 30 minutes at 23,300 g at 4° C. The insoluble pelletrecovered from centrifugation of the lysate is washed with 20 mMsodiumphosphate/500 mM NaCl, pH7.4. The pellet may optionally be furtherwashed with water, and suspended in a suspension solution as furtherdescribed herein. Other methods are described in WO2012/142515.

Proteins may be purified by isolation/purification methods for proteinsgenerally known in the field of protein chemistry. Non-limiting examplesinclude extraction, recrystallization, salting out (e.g., with ammoniumsulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration,adsorption chromatography, ion exchange chromatography, hydrophobicchromatography, normal phase chromatography, reversed-phasechromatography, gel filtration, gel permeation chromatography, affinitychromatography, electrophoresis, countercurrent distribution or anycombinations of these. After purification, proteins may be exchangedinto different buffers and/or concentrated by any of a variety ofmethods known to the art, including, but not limited to, filtration anddialysis. Methods for expressing fusion proteins in E. coli are alsoprovided in WO2012/142515.

The purified protein may be 85%, 95%, 98% or 99% pure. Regardless of theexact numerical value of the purity, the protein may be sufficientlypure for use as a pharmaceutical product.

Exemplary Uses

In one aspect, the application provides proteins, e.g., fusion proteins,comprising a fibronectin based scaffold moiety, useful in the treatmentof disorders. The diseases or disorders that may be treated will bedictated by the binding specificity of the fibronectin based scaffoldmoiety. As described herein, fibronectin based scaffold moieties may bedesigned to bind to any target of interest. Exemplary targets include,for example, TNF-alpha, VEGFR2, PCSK9, IL-23, EGFR and IGF1R. Merely asan example, fibronectin based scaffold moieties that bind to TNF-alphamay be used to treat autoimmune disorders such as rheumatoid arthritis,inflammatory bowel disease, psoriasis, and asthma. Fusion proteinsdescribed herein may also be used for treating cancer.

The application also provides methods for administering proteins to asubject. In some embodiments, the subject is a human. In someembodiments, the proteins are pharmaceutically acceptable to a mammal,in particular a human. A “pharmaceutically acceptable” compositionrefers to a composition that is administered to an animal withoutsignificant adverse medical consequences. Examples of pharmaceuticallyacceptable compositions include compositions, e.g., comprisingfibronecting based scaffold moiety, that are essentially endotoxin orpyrogen free or have very low endotoxin or pyrogen levels.

SEQUENCES WT ¹⁰Fn3 Domain:VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPS Q (SEQ ID NO: 1)¹⁰Fn3 Domain of SEQ ID NO: 1 (with D97E)VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIEKPS Q (SEQ ID NO: 2)WT ¹⁰Fn3 Domain Core Sequence version 1:LEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINY (SEQ ID NO: 3)WT ¹⁰Fn3 Domain Core Sequence version 2 :EVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 4)WT ¹⁰Fn3 Domain Core Sequence version 3:VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 5)WT ¹⁰Fn3 Domain Core Sequence version 4:VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTE (SEQ ID NO: 6)WT ¹⁰Fn3 Domain Core Sequence version 5:VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEI (SEQ ID NO: 7)WT ¹⁰Fn3 Domain Core Sequence version 6:VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEID (SEQ ID NO: 8)¹⁰Fn3 Domain Core Sequence version 7 (version 6 with D97E):VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIE (SEQ ID NO: 9)WT ¹⁰Fn3 Domain Core Sequence version 8:VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDK (SEQ ID NO: 10)¹⁰Fn3 Domain Core Sequence version 9 (version 8 with D97E):VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIEK (SEQ ID NO: 11)WT ¹⁰Fn3 Domain Core Sequence version 10:VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKP (SEQ ID NO: 12)¹⁰Fn3 Domain Core Sequence version 11 (version 10 with D97E):VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIEKP (SEQ ID NO: 13)WT ¹⁰Fn3 Domain Core Sequence version 12:VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIDKPS (SEQ ID NO: 14)¹⁰Fn3 Domain Core Sequence version 13 (version 12 with D97E):VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEIEKPS (SEQ ID NO: 15)WT ¹⁰Fn3 Domain with D8OE SubstitutionVSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGESPASSKPISINYRTEIDKPS Q (SEQ ID NO: 16)Degenerate WT ¹⁰Fn3 Domain Core Sequence:VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRT (SEQ ID NO: 17)VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRTE (SEQ ID NO: 18)VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRTEI (SEQ ID NO: 19)VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRTEID (SEQ ID NO: 20)VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRTEIE (SEQ ID NO: 21)VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRTEIDK (SEQ ID NO: 22)VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRTEIEK (SEQ ID NO: 23)VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRTEIDKP (SEQ ID NO: 24)VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRTEIEKP (SEQ ID NO: 25)VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRTEIDKPS (SEQ ID NO: 26)VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRTEIEKPS (SEQ ID NO: 27)VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRTEIDKPSQ (SEQ ID NO: 28)VSDVPRDLEVVAA(X)_(u)LLISW(X)_(v)YRITY(X)_(w)FTV(X)_(x)ATISGL(X)_(y)YTITVYA(X)_(z)ISINYRTEIEKPSQ (SEQ ID NO: 29)MGVSDVPRDL (SEQ ID NO: 30) GVSDVPRDL (SEQ ID NO: 31)X_(n)SDVPRDL (SEQ ID NO: 32) X_(n)DVPRDL (SEQ ID NO: 33)X_(n)VPRDL (SEQ ID NO: 34) X_(n)PRDL (SEQ ID NO: 35)X_(n)RDL (SEQ ID NO: 36) X_(n)DL (SEQ ID NO: 37) MASTSG (SEQ ID NO: 38)EIEK (SEQ ID NO: 39) EGSGC (SEQ ID NO: 40) EIEKPCQ (SEQ ID NO: 41)EIEKPSQ (SEQ ID NO: 42) EIEKP (SEQ ID NO: 43) EIEKPS (SEQ ID NO: 44)EIEKPC (SEQ ID NO: 45) HHHHHH (SEQ ID NO: 46) EIDK (SEQ ID NO: 47)EIDKPCQ (SEQ ID NO: 48) EIDKPSQ (SEQ ID NO: 49)MPAPTDERPTNETPSSLLISWTPPRVQITGYIIRYGPVGSDGRVKEFTVPPSVSSATITGLKPGTEYTISVIALKDNQESEPLRGRVTTGG (FibconB; SEQ ID NO: 50)TPSS (SEQ ID NO: 51) TPPRVQI (SEQ ID NO: 52) VGSDGR (SEQ ID NO: 53)PSVS(SEQ ID NO: 54) GLKPG (SEQ ID NO: 55) KDNQESEP(SEQ ID NO: 56)LDAPTDLQVTNVTDTSITVSWTPPSATITGYRITYTPSNGPGEPKELTVPPSSTSVTITGITPGVEYVVSVYALKDNQESPPLVGTCTT (SEQ ID NO: 57)LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 58)TEDS (SEQ ID NO: 59) TAPDAAF (SEQ ID NO: 60) SEKVGE (SEQ ID NO: 61)GSER (SEQ ID NO: 62) GLKPG (SEQ ID NO: 63) KGGHRSN (SEQ ID NO: 64)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 65)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 66)VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 67)EPRSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 68)EPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 69)EPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 70)EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 71)EPRSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 72)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 73)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 74)EPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 75)EPKSSDKTHTSPPSPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 76)EPKSSDKTHTSPPSPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLGSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 77)EPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLGSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 78)ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVKFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 79)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 80)EPKSSDKTHTCPPCPAPELLGGPSVFLAPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 81)EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGK (SEQ ID NO: 82)EPKSSDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 83)EPKSSDKTHTSPPSPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALGSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 84)EPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALGSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 85)EPKSSDKTHTCPPCPAPEAGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPASIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 86)DKTHTCPPCPAPELLG (SEQ ID NO: 87)EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 88)EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 89)EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 90)DKTHTCPPCPAPELLGGPS (SEQ ID NO: 91) DKTHTCPPCPAPELLGGSS (SEQ ID NO: 92)EPKSSDKTHTCPPCP (SEQ ID NO: 93)

The following representative Examples contain important additionalinformation, exemplification and guidance which can be adapted to thepractice of this invention in its various embodiments and theequivalents thereof. These examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit its scope.

EXAMPLES Example 1: High-Throughput Screening of Buffer Conditions forRefolding Denatured Proteins

In order to identify effective refolding conditions for ¹⁰Fn3/Fc fusionproteins produced in E. coli, an automated liquid handling platform wasused to execute protein refolding by dilution in triplicate in 96-wellplates.

The biomass of a ¹⁰Fn3/Fc protein was produced in a 10 L fermentor. Thebiomass was harvested and the IBs were recovered and washed bycentrifugation before being frozen.

The screening study looked at protein concentration, resolubilizationbuffer, refold pH, temperature, aggregation suppressing excipients, andredox excipients. JMP Design of Experiments (DoE) software was used todesign the screening study and analyze data. Data were gathered using aplate reader and SE-HPLC. The data suggested a resolubilization bufferaround pH 8 with Guanidine and a refold buffer with Arginine to suppressaggregation, and a Glutathione redox system, around pH 10, favoredformation of soluble protein. In addition, these conditions showedprotein around the correct molecular weight in solution, indicatingdisulfide bond formation, required to form the ¹⁰Fn3/Fc homodimer.

Scale up of the dilution refold to 50 mL, 100 mL and 200 mL final refoldvolumes using the above-identified conditions were performed using acalibrated pump and mixing. A variable and heavy precipitation event wasobserved in all cases and a low recovery of protein was observed. Onlyaround 10 to 20% of the protein was recovered in solution and found tobe at the appropriate molecular weight. For a subset of the bench scaledilution refolds, a majority of the protein recovered in solution wasfound to be at a smaller molecular weight corresponding to ¹⁰Fn3/Fcmonomer and indicating that the disulfide bonds did not form. With thesedata, it was determined that alternative methods of refolding ¹⁰Fn3/Fcmolecules should be evaluated.

Example 2: Effect of pH on Refolding Proteins from IBs

This Example describes that the efficiency of refolding denatured Fcfusion proteins varies with the pH of the buffer used for refolding theproteins, and that refolding is more efficient at higher pH than atlower pH.

The refold efficiency of an Fc fusion protein under different conditionswas analyzed using Sephadex G25 chromatography. Briefly, a column isconditioned with refold buffer, following which solubilized IBs areadded to the column and the protein from the IBs is recovered by passingthe same refold buffer as that used for conditioning the column over thecolumn.

The Fc fusion protein used was a ¹⁰Fn3/Fc molecule having the followingamino acid sequence:

(SEQ ID NO: 94; the ¹⁰Fn3 sequence is shown in italics)MGVSDVPRDLEVVAATPTSLLISWVPPSDDYGYYRITYGETGGNSPVQEFTVPIGKGTATISGLKPGVDYTITVYAVEFPWPHAGYYHRPISINYRTEIEPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

The ¹⁰Fn3/Fc protein was expressed in E. coli and the IBs werecollected. The IBs were solubilized at a weight (grams):volume (ml)ratio of 1:20 in 50 mM Tris pH 8.0, 6.0 M GuHCl, 0.2 mM TCEP. Thesolubilization reaction was mixed until complete solubilization of IBsoccurred. The solubilization reaction was then mixed with a 2× volume of50 mM Tris, 0.4M arginine pH 10.4, and 10 ml thereof was loaded onto2.5×10 cm Sephadex G25 columns at 150 cm/hr. The G25 Sephadex columnswere conditioned with refold buffer consisting of 0.4 M Arginine and 50mM Tris pH 8.5, 9.0 or 10.4 by loading a 10% column volume equivalent ofrefold buffer on the column at 150 cm/hr until stable baseline pH wasachieved. Absorbance of the eluate was monitored at 280 nm and the majorabsorbance peak of each column was collected for further analysis.

The chromatograms of the experiments conducted at pH 8.5, 9.0 and 10.4are shown in FIGS. 2A, B and C, respectively. Aggregate and precipitatedprotein can be visualized in the chromatograms as peak shifts or as NaOHstrip peaks. The chromatogram of the refold experiment conducted at pH8.5 (FIG. 2A) shows a significant shift as well as a NaOH strip peak,whereas the chromatograms of the refold experiments conducted at pH 9.0and 10.4 (FIGS. 2B and C, respectively) show good peak symmetry and noNaOH strip peak, indicating less aggregation and protein precipitation,and higher solubility of the proteins at pH 9.0 and 10.4 than at pH 8.5.As solubility is used as an indicator of proper protein folding in theseexperiments, the results indicate that refolding denatured protein at pH9.0 and 10.4 is more efficient than at pH 8.5.

Efficiency of refolding at the different pH values was also analyzedwith SDS PAGE. This method shows soluble aggregated protein, which isalso indicative of protein misfolding. 10 μl of protein from the majorabsorbance peaks that were eluted from the G25 Sephadex columnsdescribed above were diluted with 20 μl of water and 10 μl LDL samplebuffer, and 30 μl of diluted sample was loaded on a 10 well 4-12%Bis-Tris Gel (Novex). The gel was run at 200V for 35 minutes. The gelwas rinsed with water for 10 minutes, stained overnight with thermogelcode blue stain and destained for 3 hours prior to scanning.

The stained gels are shown in FIGS. 3A and B. These indicate thatsignificant soluble aggregate is present in the refold done at pH 9.0,whereas at pH 10.4, less soluble aggregate is present and more dimer ispresent, suggesting that refolding and dimer formation is more efficientat higher pH.

Example 3: A Reducing Agent is not Necessary During Refolding

Certain methods for refolding proteins comprising disulfide bonds usetwo refolding steps: a first refolding step during which a reducingagent, e.g., TCEP, is used to maintain a reduced conformation of thecysteines, to first refold the monomers; and a second refolding stepduring which disulfide bonds are formed, to dimerize the properly formedmonomers. In this Example the necessity of including a reducing agentduring a first refold step was investigated, and the results show that areducing agent is not necessary for obtaining efficient refolding of adenatured protein.

The same experiment as that described in Example 2 was performed withthe refold buffer at pH 10.4. The protein of the major absorbance peakwas then incubated at room temperature for 30 minutes or 4 hours in thesame buffer (i.e., in the absence of TCEP), prior to being loaded ontoan SDS-PAGE gel. The gel was loaded and run and stained essentially asdescribed in Example 2.

The stained SDS-PAGE gel is shown in FIGS. 4 A and B. The results showthat dimer formation does not occur in the absence of TCEP even afterthe longer (4 hours) incubation time. This finding suggests that it isnot necessary to include a reducing agent, e.g., TCEP, during the firstrefolding step. In addition, eliminating TCEP from the ¹⁰Fn3/Fcrefolding procedures resulted in significant reduction of CH3 open loopcontent of ¹⁰Fn3/Fc preparations.

Example 4: The First Refolding Step Occurs Rapidly

This Example shows that refolding denatured protein during the firstrefolding step occurs rapidly, and therefore a long incubation timeprior to the second refold (oxidizing) step is not necessary.

If proper refolding of monomer is required prior to correct dimerformation and the rate of refolding of the monomer is slow, it may benecessary to conduct the monomer refolding step (i.e., refold step 1)for a long time. The results of the G25 refold experiments described inExamples 1 and 2, however, appear to show that proper folding of¹⁰Fn3/Fcs can be accomplished using an almost instantaneous removal ofdenaturant (i.e., a short refold step 1). The instant Example confirmsthis observation by testing different refold step 1 incubation times.

The same experiment as that described in Example 2 was performed withthe refold buffer at pH 10.4. The protein of the major absorbance peakwas then incubated at room temperature for 0, 1 hour or 2 hours eitheras eluted or after diluting it 1:1 with 50 mM Tris, 0.4M Arginine pH10.4 refold buffer. After the incubation, Glutathione at a concentrationof 1:0.2 mM oxidized:reduced was then added to samples. The reactionswere then incubated for another 3 hours at room temperature. SDS-PAGEwas then performed essentially as described in Example 2.

The stained gels, which are shown in FIG. 5, indicate that similarlevels of dimer formation occurred in all samples regardless of theincubation time of the samples (corresponding to refold step 1),suggesting that refolding occurs rapidly after the denaturing agent isremoved. Although refold occurred in both the samples directly elutedfrom the column and those diluted 1:1 with refold buffer, dimerformation appears to be more efficient in the diluted samples.

Example 5: Solubilization of IBs at High pH in the Absence of GuanidineHydrochloride

This Example shows that IBs can be solubilized in a buffer having highpH in the absence of a denaturing agent, such as guanidinehydrochloride.

Solubilization efficiency of ¹⁰Fn3/Fc IBs in solubilization buffer withpH values from 10.4 through 12.5 was explored. IBs of the same proteinas that in the previous Examples were added to a 20× volume of 50 mMTris, 0.4M Arginine pH 10.4. Mixing was initiated, and the pH was slowlyadjusted with 12M NaOH until all IBs were solubilized. The pH of thesolution at solubilization was pH 12.2.

Later experiments explored reducing the pH of the solubilization usingProline and Glycine as additives. The only condition at lower pH thatwas successful in solubilizing IBs in a reasonable time frame was 50 mMTris, 0.5M Arginine, 0.1M Proline pH 10.7. Solubilization took 20minutes in this solution. Thus, at lower pH values, additives wererequired to fully solubilize the IBs, and the solubilization tooksignificantly longer than at higher pH. Solubilization at pH 12.2 for3-5 minutes was found to solubilize essentially all IBs in the reaction.

Example 6: Characterization of the Protein Folded State at Different pHs

This Example shows that at least partial tertiary structure of a¹⁰Fn3/Fc protein is maintained during solubilization of IBs at high pH,but not in a 6M Guanidine solution.

To determine whether a ¹⁰Fn3/Fc protein retains a secondary and tertiarystructure during solubilization at high pH, Far and Near UV CircularDichroism (CD) was used.

A ¹⁰Fn3/Fc protein comprising a human IgG1 Fc linked to the N-terminusof a ¹⁰Fn3 moiety binding to a different target from that bound by themolecule used in Examples 2-5 was expressed in E. coli and IBs wereisolated. The Fc has the following amino acid sequence:

(SEQ ID NO: 95) MGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP.

As Arginine is not compatible with CD, a solubilization buffer differentfrom that in Example 5 was used. A purified high concentration sample of¹⁰Fn3/Fc protein was diluted into the following buffers at a finalconcentration of 1 mg/ml: 50 mM phosphate pH 7.2; 50 mM Tris pH 8.0; 50mM carbonate pH 10.2; 50 mM phosphate pH 12.2; 50 mM Tris pH 8.0+6MGuHCl. A sample of protein diluted into 50 mM phosphate pH 12.2 wasadjusted back to pH 7.5 using hydrochloric acid. The samples weresubjected to Far and Near UV CD.

The results, which are shown in FIGS. 6A and B, indicate that at high pHsome tertiary structure is maintained while with 6M Guanidine all thesecondary and tertiary structure is lost (see FIGS. 6A and B). At pH10.4 most of the secondary and tertiary structure is maintained.Bringing the protein from pH 12.2 to pH 7.5 showed partial refolding ofthe molecule. Thus, the results indicate that a denatured protein thatis solubilized in a solubilizing buffer at high pH but in the absence ofa denaturing agent maintains some tertiary structure, and that this mayfacilitate refolding of the protein relative to asolubilization/refolding method using a denaturing agent, such asguanidine hydrochloride.

Example 7: High pH IB Solubilization and Refold of ¹⁰Fn3/Fc Proteins

This Example describes a method for solubilizing and refolding denaturedproteins in the absence of a denaturing agent. The method was used tosolubilize and refold several different ¹⁰Fn3/Fc proteins, some of whichwere in the ¹⁰Fn3-Fc format and others in the Fc-¹⁰Fn3 format.

All steps were performed at room temperature.

15 grams of IBs were suspended in 30 mls of Milli Q water. Thesuspension was mixed until a uniform appearance was observed and thenplaced aside.

Solubilization buffer was prepared by adding 1.398 ml of 12.5M NaOH to300 mls of 50 mM Tris, 0.4M Arginine pH 10.4. Final pH after additionwas 12.2.

Refold buffer was prepared as follows. A total of 0.915 g of oxidizedglutathione and 0.0915 g of reduced glutathione was added to 900 ml of50 mM Tris pH, 0.4 M Arginine pH 10.4. A total of 1.701 ml of 12.0M HClwas spiked into the refold buffer+glutathione.

Table 1 provides the amount of HCl to be added to various volumes ofRefold buffer to achieve a pH around 10.4.

TABLE 1 volumes of 12M HCl to add to refold buffer to obtain a pH ofabout 10.4 Volume of 12.0M Volume of Volume of refold HCl to add torefold solubilization buffer buffer at 1:3 dilution buffer prior todilution 1 ml 3 ml 5.67 μl  250 ml 750 ml 1.42 ml 750 ml 2.25 L 4.25 ml1 L 3 L 5.67 ml

Solubilization was conducted as follows. Mixing of inclusion bodysuspension was initiated on a stir plate. The 300 ml of solubilizationbuffer were added to the mixing IB suspension and a timer was started.

Mixing of the 900 ml of refold buffer was initiated at 2 minutes.

After 2.5 minutes of mixing the IB suspension with the solubilizationbuffer, the mixing solubilization solution (or solubilization reaction)appeared transparent and there were no intact IBs present in thesolution.

The solubilization reaction was then poured into the mixing refoldbuffer as quickly as possible (<5 seconds). Mixing was continued for 30seconds after the solubilization reaction was added so that completemixing of the two solutions was accomplished.

The refold reaction was then removed from the stir plate and allowed tosit static at room temperature for 1 hour. After 1 hour the refoldreaction was placed back on the stir plate and mixing was againinitiated. Once mixing occurred, 380 ml of a 0.1M HCl solution was addedto the refold reaction as quickly as possible (<5 seconds). Refoldingwas accomplished at this point, and the refolded protein can be loadedonto protein A for purification.

Example 8: Refolding of a ¹⁰Fn3/Fc Protein

This Example describes a method for solubilizing and refolding denaturedproteins in the absence of a denaturing agent.

Cell paste from the induction phase of fermentation of an E. coliculture expressing the ¹⁰Fn3/Fc protein of Example 6 was removed from−80° C. storage and suspended in 20 mM Sodium Phosphate pH 6.2, 250 mMSodium Chloride, 5 mM EDTA at a ratio of 1:10 (W/W solids/buffer) usingan UltraTurrax. The suspended material was passed through amicrofluidizer twice at a psi of 18,000. The disrupted suspension wascentrifuged at 10,000×g for 30 minutes to isolate the insolublefraction. This fraction was resuspended, washed, and isolated twice.Wash Buffer was 20 mM Sodium Phosphate pH 6.2, 250 mM Sodium Chloride, 5mM EDTA, 1% Triton X-100. The isolation was performed via centrifugation10,000×g. The remaining insoluble fraction was then washed twice more inDI water. Isolation was performed via centrifugation at 10,000×g for 30minutes. The isolated insoluble fraction (IBs) was stored at −20° C.

The ¹⁰Fn3/Fc protein fusion in the IBs was refolded as follows. FrozenIBs were thawed in RODI water. Once the IB solution was fullyhomogenous, the IBs were resolubilized by dilution with 50 mM Tris 0.4MArginine pH 12.2 with stirring. This solution was left stirring for 2-5minutes, until fully dissolved and no large particles were visible.After solubilization, the sample was diluted further with the additionof 50 mM Tris 0.4M Arginine pH 10.4, with the extra addition of 12M HClto bring the pH of the solution to 10.4. To refold the protein, a redoxreaction was performed using a 1 mM: 0.2 mM oxidized to reduced ratio ofGlutathione. Redox reaction was left for 1 hour at room temperature.After 1 hour, the pH of the reaction was dropped to about pH 8.0 withthe addition of 0.3× volume of 1M HCl.

Example 9: Effect of Protein Concentration on Efficiency of High pHSolubilization and Refolding

This Example shows that the high pH method for solubilizing andrefolding denatured protein can be used with concentrations of proteinsof up to at least 7 mg/ml.

The high pH solubilization method described Examples 7 and 8 was usedfor refolding ¹⁰Fn3/Fc protein (used in Examples 5 and 7) at 1.75, 3.5and 7 mg/ml. Protein concentration was determined at the solubilizationstage and adjusted for the later dilution. The concentration wasmeasured by absorbance at 280 nm. 10 or 20 μg of refolded protein weresubjected to SDS-PAGE analysis. The gel, which is shown in FIG. 7,indicates that dimer formation occurred at each of these concentrations.Therefore, concentrations of protein up to at least 7 mg/ml at thesolubilization stage can be refolded using this method. Dimer formationwas, however, more efficient at lower refold concentrations.

Example 10: Control of Deamidation that Occurs at High pH

A concern with using high pH to solubilize and refold ¹⁰Fn3/Fcs isdeamidation of the protein. This Example shows that limiting the time ofincubation at high pH for solubilization reduces deamidation.

In early experiments, solubilization at pH 12.2 as described in Examples7 and 8 was conducted for one hour at room temperature and the refold(at pH 10.4) was incubated overnight at room temperature. The refoldshowed Iso-asp formation at a level of 28.1% and 17.8% deamidation bylys-C digestion followed by peptide map analysis.

To try to reduce deamidation levels, a solubilization and refoldperformed as in Examples 7 and 8 with the ¹⁰Fn3/Fc protein of Example 5was conducted with a solubilization time of 5 minutes and a refold of18-20 hours at pH of 10.4. The refolded protein showed only 5.6%iso-asp, 3.7% deamidation and 2.7% imide by lys-C peptide map.

Thus, by minimizing exposure to both pH 12.2 during the solubilizationand pH 10.4 in the refold buffer, deamidation was minimized. The high pHsolubilization and refolding method may provide more control overrefolding time and therefore deamidation, considering that chemicaldenaturant removal is not required and simply reducing the pH willreduce deamidation.

Example 11: Determination of pH of Solubilization and Refold Reactions

This Example describes the pH of the solubilization and refold reactionswhen performing the solubilization and refold method described inExamples 7 and 8.

The ¹⁰Fn3/Fc protein used in Examples 6 and 7 was expressed in E. coliand IBs were obtained. 0.5 grams of IBs were suspended in 1 ml of MilliQ water. The suspension was mixed until a uniform appearance wasobserved and then placed aside.

Solubilization buffer was prepared by adding 46.6 μL of 12.5M NaOH to 10mls of 50 mM Tris, 0.4 M Arginine pH 10.4. The final pH of thissolubilization buffer was 12.3.

A total of 0.0305 g of oxidized glutathione and 0.003 g of reducedglutathione was added to 30 ml of 50 mM Tris, 0.4 M Arginine pH 10.4.The final pH of this refold buffer was 10.4.

A total of 56.7 μl of 12.0 M HCl was spiked into the refold buffercontaining glutathione.

The entire 10 ml of solubilization buffer was added to the IBsuspension. After 3 minutes, the mixing solubilization solution appearedtransparent and there were no intact IBs present in the solution. The pHof this solubilization reaction was 12.5.

The solubilization reaction was then poured into the mixing refoldbuffer as quickly as possible (less than 5 seconds). Mixing wascontinued for 30 seconds after the solubilization reaction was added sothat complete mixing of the two solutions was accomplished. The pH ofthis refold reaction was 10.3.

All steps of the method described in this Example were performed at roomtemperature.

Thus, the pH of the solubilization and refold reactions vary slightlyfrom the pH of the solubilization and refold buffers, respectively. ThepH of the solubilization reaction was pH 12.05, compared to the buffer,which was 12.3.

The pH of the refold reaction is also affected by the protein insolution. The pH of the refold reaction was measured at 10.3. Fcdimerization should be effective for any pH above 10.0, although a pH ofas low as 9.0 can be used, although at such a pH protein aggregation mayoccur.

Example 12: Comparison of Target Binding of Refolded and MammalianExpressed ¹⁰Fn3/Fc Protein

This experiment shows that a ¹⁰Fn3/Fc protein that was expressed in E.coli and refolded as described herein binds similarly to its targetprotein relative to the same protein that was expressed in mammaliancell culture.

A ¹⁰Fn3-¹⁰Fn3-Fc (i.e., a bispecific molecule having two ¹⁰Fn3 entitiesbinding to a bispecific molecule having two ¹⁰Fn3 entities binding totwo different targets) protein was expressed both in E. coli andrefolded essentially as described in Example 8 and 9. The same proteinwas also expressed in mammalian cells HEK293-6E. Binding of both ¹⁰Fn3entities to their target was determined by SPR. The following format wasused. Protein A was covalently linked to a chip. 1.5 nM ¹⁰Fn3-¹⁰Fn3-Fcwas captured on the protein A. Binding to one of the two target proteinswas measured by SPR. 0.15-5 nM of the target was used.

The results, which are shown in FIGS. 8A and B, indicate that the¹⁰Fn3-¹⁰Fn3-Fc protein expressed in E. coli and refolded has similartarget binding kinetics to those of the ¹⁰Fn3-¹⁰Fn3-Fc protein that wasexpressed in mammalian cells. Thus, ¹⁰Fn3/Fc proteins expressed in E.coli and refolded as described herein is at least sufficiently refoldedto allow binding to its target.

Example 13: Comparison of Inhibition of Biological Activity of Refoldedand Mammalian Expressed ¹⁰Fn3/Fc Protein

This Example shows that a ¹⁰Fn3/Fc protein that was expressed in E. coliand refolded as described herein has similar biological activityrelative to the same protein that was expressed in mammalian cellculture.

In this experiment mice are injected with the bispecific ¹⁰Fn3-¹⁰Fn3-Fcof Example 12 made either in E. coli or in mammalian cells, and thelevel of activity of the ¹⁰Fn3-¹⁰Fn3-Fc molecule was determined bydetermining the level of inhibition of the biological activity of thetarget. The mammalian protein was produced in a mammalian shake flaskculture or in a mammalian bioreactor. For comparison purposes, theexperiment also included an antibody to one of the two targets and a theadnectin binding to one of the targets alone (i.e. a mono-adnectin).Different concentrations of each were tested. Three different biologicalactivities of the target protein were tested: two of the activitiesconsisted of the induced secretion of two different cytokines and thethird biological activity of the target protein was stimulation ofsignal transduction.

Inhibition of the secretion of one cytokine is shown in FIG. 9. Theresults indicate that the ¹⁰Fn3-¹⁰Fn3-Fc protein expressed in E. coliand refolded as described herein inhibit cytokine secretion induced bythe target to a similar level. A similar result was seen when measuringthe level of inhibition of secretion of the second cytokine (FIG. 10).

Inhibition of signal transduction is shown in FIG. 11. The resultsindicate that the ¹⁰Fn3-¹⁰Fn3-Fc protein expressed in E. coli andrefolded as described herein inhibit signal transduction induced by thetarget to a similar level.

Thus, these results taken together with those of Example 12 indicatethat the ¹⁰Fn3-¹⁰Fn3-Fc protein expressed in E. coli and refolded asdescribed herein is at least sufficiently refolded to have biologicalactivity that is similar to that of the same protein expressed in amammalian cell culture system.

The entire disclosures of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books,GENBANK® Accession numbers, SWISS-PROT® Accession numbers, or otherdisclosures) in the instant patent application, including theBackground, Detailed Description, Brief Description of the Drawings, andExamples, are hereby incorporated herein by reference in their entirety.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any that are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

The invention claimed is:
 1. A method for refolding a denatured protein,comprising suspending a denatured protein in a suspension solution toobtain a composition comprising suspended denatured proteins; combiningthe composition comprising suspended denatured proteins with asolubilization buffer having a pH in the range of 11.5 to 12.8 tothereby obtain a composition comprising solubilized denatured proteins;and combining the composition comprising solubilized denatured proteinswith a refold buffer having a pH in the range of 10 to 10.9 to therebyobtain a composition comprising refolded proteins; wherein the methoddoes not include the use of a denaturing agent.
 2. The method of claim1, wherein the suspension solution consists of water.
 3. The method ofclaim 1, wherein the denatured proteins are suspended in suspensionsolution at a ratio of weight (g) of denatured proteins:volume (ml) ofsuspension solution of 1:1-3.
 4. The method of claim 1, wherein thecomposition comprising suspended denatured proteins is combined withsolubilization buffer at a ratio of weight (g) of denaturedproteins:volume (ml) of solubilization buffer of 1:10-30.
 5. The methodof claim 1, wherein the composition comprising solubilized denaturedproteins is combined with refold buffer at a ratio of volume ofsolubilization buffer:volume of refold buffer of 1:1-5.
 6. The method ofclaim 1, wherein the denatured proteins are suspended in suspensionsolution at a ratio of weight (g) of denatured proteins:volume (ml) ofsuspension solution of 1:1-3; the composition comprising suspendeddenatured proteins is combined with solubilization buffer at a ratio ofweight (g) of denatured proteins:volume (ml) of solubilization buffer of1:10-30; and the composition comprising solubilized denatured proteinsis combined with refold buffer at a ratio of volume of solubilizationbuffer:volume of refold buffer of 1:1-5.
 7. The method of claim 1,wherein the suspended denatured proteins and the solubilization bufferare combined for 1-10 minutes prior to being combined with the refoldbuffer.
 8. The method of claim 1, wherein the composition comprising thesolubilized denatured proteins is combined with the refold buffer for5-60 minutes.
 9. The method of claim 1, wherein the solubilizationbuffer comprises Arginine.
 10. The method of claim 1, wherein the refoldbuffer comprises Arginine.
 11. The method of claim 1, wherein the refoldbuffer comprises an oxidizing agent.
 12. The method of claim 1, whereinthe method does not comprise first suspending the denatured protein in asuspension solution.
 13. The method of claim 1, wherein the denaturedproteins are in the form of inclusion bodies (IBs).
 14. The method ofclaim 1, wherein the protein comprises an Fc region.
 15. The method ofclaim 14, wherein the protein comprises a binding domain thatspecifically binds to a target protein, and wherein the binding domainis an alternative scaffold binding domain.
 16. The method of claim 15,wherein the alternative scaffold binding domain is a fibronectin basedscaffold domain.